Peptide compounds and methods of production and use thereof

ABSTRACT

Peptide compounds based on the CAP37 protein are disclosed, along with methods for treating various infections, wounds, and conditions, and methods of promoting healing and acceptance of grafts, using compositions containing these peptide compounds.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

This application claims benefit under 35 USC §365(c) of InternationalApplication No. PCT/US2013/072884, filed Dec. 3, 2013; which claimsbenefit of US provisional application U.S. Ser. No. 61/812,584, filedApr. 16, 2013, and US provisional application U.S. Ser. No. 61/813,527,filed Apr. 18, 2013. The entire contents of the above-referenced patentapplications are hereby expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Public HealthService Grant Number R01EY0155534 awarded by the National Eye Institute(NEI) of the National Institutes of Health (NIH) and under Grant Number5U01A1075391 awarded by the National Institute of Allergy and InfectiousDiseases (NIAID) of the NIH. The government has certain rights in theinvention.

BACKGROUND

Effective treatment of Gram negative bacterial infections has suffereddue to a dearth of new antibiotics in the pharmaceutical pipeline. Onlyone novel antibacterial has been approved since 2006. Thus there is aclear unmet need for such therapies. Antibiotic resistance is one of thegreatest threats to global human health. Thus, therapies with novelmechanisms of action that could circumvent resistance are highlydesired. For example, sepsis or septic shock kills more than 200,000people per year in the U.S. alone. Optimally, a sepsis therapeutic wouldserve as an antibiotic, as well as bind and neutralize the toxic effectsof endotoxin (a.k.a., lipopolysaccharide, or LPS). The morbidity andmortality that results from severe infection in the U.S. alone isestimated at an annual cost of many billions of dollars. In the EuropeanUnion the lost productivity and health care costs due to multi-drugresistant infections is estimated at 1.5 billion euros per year. Newbioactive peptides that are effective against Gram negative infectionsand exhibit no or low toxicity would be a significant contribution tothis global health crisis.

CAP37 (cationic antimicrobial protein of M_(r) 37 kDa) was originallyidentified as a component of the oxygen-independent killing mechanism ofthe human neutrophil (PMN) and was demonstrated to have strongbactericidal activity against Gram negative bacteria includingSalmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa.Distinct from its effect on bacteria, the native CAP37 protein haspotent regulatory effects on host cells. It is an effective regulator ofcells of the mononuclear phagocytic system such as monocytes, microglia,and macrophages. It also regulates certain corneal epithelial,endothelial, and smooth muscle cell functions.

Structure-function analysis of CAP37 previously enabled the delineationof an antibacterial domain of the CAP37 protein, which was identified asresiding in residues 20 through 44 of the native molecule. A peptidecomprising this 25 amino acid sequence (i.e., CAP37 (20-44) SEQ ID NO:7) mimicked the antimicrobial activity of the native molecule andextended its range of activity to encompass Staphylococcus aureus andEnterococcus faecalis, two Gram positive bacteria. The bactericidalactivity of the peptide was pH dependent, with maximum activity obtainedbetween pH 5.0 and 5.5. Derivatives of the natural peptide sequence of20-44, wherein either of the cysteine residues at position 26 or 42 werereplaced with serine residues (CAP37 (20-44)_(ser26) and CAP37(20-44)_(ser42)—SEQ ID NO:1 and 4, respectively), were also produced.However, commercialization of a peptide as a first-in-classanti-infective requires the ability to, for example, (1) scale-up thesynthesis of the peptide, (2) achieve purity of preferably >90%, and (3)retain activity. The 20-44 peptide and previously-known substitutedversions thereof could not be produced with these features. It is tonovel peptide compounds that possess such properties and which comprisevarious portions of CAP37 proteins and derivatives thereof, as well asto methods of use of these compounds, that the presently disclosedinventive concepts is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

Several embodiments of the present disclosure are hereby illustrated inthe appended drawings. It is to be noted however, that the appendeddrawings only illustrate several typical embodiments and are thereforenot intended to be considered limiting of the scope of the presentdisclosure. Further, in the appended drawings, like or identicalreference numerals may be used to identify common or similar elements,and not all such elements may be so numbered. The figures are notnecessarily to scale, and certain features and certain views of thefigures may be shown exaggerated in scale or in schematic in theinterest of clarity and conciseness.

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows that BCC02-5RMP (SEQ ID NO:21) has antimicrobial activityin mouse bacterial keratitis. A circular wound was created on the mousecornea by removing the epithelium and infected with 10⁵ colony formingunits (CFU) of P. aeruginosa (ATCC® 27853™ (American Type CultureCollection, Manassas, Va.)). Infected wounds were treated with saline(0.9% sodium chloride, vehicle control) or saline containing indicatedconcentrations of peptide every 15 minutes for 2 hours, then every 30minutes for 3 hours on the first day. Infected wounds were treated twiceon the second day and once on the third day. Mice were killed at 48hours post-infection, and CFU/eye were quantified. The means wereplotted and are representative of 5 to 9 mice per group. Mann Whitneytest was performed for each group as compared to the saline controlgroup. *P=0.014. In compound names such as BCC02-5RMP used herein, “R”represents arginine, and “MP” represents a small PEG molecule as definedelsewhere herein (such as, for example but not by way of limitation,“AEEP”).

FIG. 2 shows that peptide 95-122 (SEQ ID NO:26) promotes corneal woundhealing in vivo. The mouse corneal epithelium was removed using theAlgerBrush II (The Alger Company, Inc., Lago Vista, Tex.), and cornealabrasions were treated at 0 hours and 16 hours with peptide 95-122 (10⁻⁵M), or were left untreated in 0.9% sodium chloride (saline vehicle)Wound healing was monitored at 0, 16, and 24 hours using fluoresceinstaining and a camera-equipped inverted microscope. Data are representedas the percentage of wound closure at 16 hours and 24 hours and areexpressed as means±SEM. The data are representative of at least 6 miceper group. *P<0.05 by unpaired t-test as compared to vehicle (0.9%sodium chloride) treated controls.

FIG. 3 shows that peptide 95-122 (SEQ ID NO:26) promotes corneal woundhealing in vivo. The mouse corneal epithelium was removed using theAlgerBrush II, and corneal abrasions were treated at 0 hours and 16hours with peptide 95-122 (10⁻⁵M), or were left untreated in 0.9% sodiumchloride (saline vehicle). Wound healing was monitored at 0, 16, and 24hours using fluorescein staining and a camera-equipped invertedmicroscope. Representative images of in vivo corneal abrasions are shownat 0, 16, and 24 hours.

FIG. 4 shows that peptide 95-122 (SEQ ID NO:26) significantly increasesIL-7, IL-15, IFN-γ, and MIP1-α. Corneas were injected intrastromallywith 0.5 μl of peptide 95-122 at a final concentration of 10⁻⁴ M, 10⁻⁵M, and 10⁻⁶ M and vehicle (saline) control. Corneas were collected andflash frozen at 24 hours and 48 hours. Corneal lysates were analyzed forcytokines using the MILLIPLEX® MAP mouse cytokine assay for IL-7(A),IL-15 (B), IFN-γ (C), MIP1-α (D) (EMD Millipore Corp., Billerica,Mass.). The means of independent experimental values are shown±SEM.***P<0.001, **P<0.01, *P<0.05 by unpaired t-test as compared to vehicletreated controls.

FIG. 5 shows that peptide 95-122 (SEQ ID NO:26) significantly increasesMIP1-β. Corneas were injected intrastromally with 0.5 μl of peptide95-122 at a final concentration of 10⁻⁴M, 10⁻⁵M, and 10⁻⁶M and vehicle(saline) control. Corneas were collected and flash frozen at 24 hoursand 48 hours. Corneal lysates were analyzed for cytokines using theMILLIPLEX® MAP mouse cytokine assay for MIP1-β (EMD Millipore Corp.,Billerica, Mass.). **P<0.01, *P<0.05 by unpaired t-test as compared tovehicle treated controls.

FIG. 6 shows that peptide 95-122 (SEQ ID NO:26) significantly increasesKC, IL-1β, and GM-CSF. Corneas were injected intrastromally with 0.5 μlof peptide 95-122 at a final concentration of 10⁻⁴M, 10⁻⁵M, and 10⁻⁶Mand vehicle (saline) control. Corneas were collected and flash frozen at24 hours and 48 hours. Corneal lysates were analyzed for cytokines usingthe MILLIPLEX® MAP mouse cytokine assay for KC (A), IL-1β (B), andGM-CSF (C) (EMD Millipore Corp., Billerica, Mass.). **P<0.01, *P<0.05 byunpaired t-test as compared to vehicle treated controls.

FIG. 7 shows that peptides 120-146QH (SEQ ID NO:29) and 120-146QR (SEQID NO:28), based on the native sequence of CAP37, mediate human cornealepithelial cell (HCEC) chemotaxis. The effect of buffer control (0.1%BSA in Gey's buffer), heparin binding-epidermal growth factor (HB-EGF,50 ng/ml), recombinant CAP37 (rCAP37, 250 ng/ml), 120-146QH (10⁻¹²M,10⁻¹⁰ M, 10⁻⁸ M, 10⁻⁶M, and 10⁻⁴M,) and 120-146QR (10⁻¹⁰ M, 10⁻⁸M,10⁻⁶M, and 10⁻⁴M) on HCEC chemotaxis was determined by the modifiedBoyden chemotaxis chamber method. HCEC chemotaxis was measured inresponse to HB-EGF, rCAP37, and peptides 120-146QH (SEQ ID NO:29) and120-146QR (SEQ ID NO:28) after incubation for 3 hours at 37° C.Chemotaxis is expressed as a percent migration. The buffer control (nochemoattractant) is arbitrarily assigned the value of 100% migration.Data are expressed as means±SEM and are calculated from 6 observationsfor each test point. **P<0.01 by Wilcoxon signed-rank test as comparedto controls.

FIG. 8 shows that peptide 120-146WH (SEQ ID NO:31) promotes cornealwound healing in vivo. The mouse corneal epithelium was removed usingthe AlgerBrush II, and corneal abrasions were treated at 0 and 16 hourswith peptide 120-146WH (10⁻⁸ M or 10⁻⁶ M), or were left untreated in0.9% sodium chloride (saline vehicle control). Wound healing wasmonitored at 0, 16, and 24 hours using fluorescein staining and acamera-equipped inverted microscope. Data are represented as thepercentage of wound closure at 16 hours and 24 hours and are expressedas means±SEM. The data are representative of at least 6 mice per group.***P<0.001,*P<0.05 by unpaired t-test as compared to vehicle (0.9%sodium chloride) treated controls.

FIG. 9 shows that peptide 120-146WH (SEQ ID NO:31) promotes cornealwound healing in vivo. The mouse corneal epithelium was removed usingthe AlgerBrush II, and corneal abrasions were treated at 0 and 16 hourswith peptide 120-146 WH (10⁻⁸ M or 10⁻⁶M), or were left untreated in0.9% sodium chloride (saline vehicle control). Wound healing wasmonitored at 0, 16, and 24 hours using fluorescein staining and acamera-equipped inverted microscope. Representative images of in vivowound closure of corneal abrasions are shown at 0, 16, and 24 hours.

FIG. 10 shows bactericidal activity of CAP37 peptides 120-146WR (SEQ IDNO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26) comparedwith gentamicin as the comparator antibiotic. Each of the test peptideswas used at 25, 12.5 6.25 and 3.12 μM. The gentamicin was used at 4μg/ml. 1×10⁶/ml Acinetobacter baumannii ATCC® BAA-747™ (American TypeCulture Collection, Manassas, Va.) were incubated with the peptides,negative buffer control (tryptone saline), and positive controlgentamicin for 60 minutes at pH 5.5. 50 μl aliquots were plated out onagar plates and incubated overnight. Colony forming units were countedafter overnight incubation and CFU/ml calculated.

FIG. 11 shows bactericidal activity of CAP37 peptides 120-146WR (SEQ IDNO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26) comparedwith gentamicin as the comparator antibiotic. Each of the test peptideswas used at 25, 12.5 6.25 and 3.12 μM. The gentamicin was used at 4μg/ml. 1×10⁶/ml Acinetobacter baumannii ATCC® BAA-747™ (American TypeCulture Collection, Manassas, Va.) were incubated with the peptides,negative buffer control (tryptone saline), and positive controlgentamicin for 60 minutes at pH 7.2. 50 μl aliquots were plated out onagar plates and incubated overnight. Colony forming units were countedafter overnight incubation and CFU/ml calculated.

FIG. 12 shows bactericidal activity of CAP37 peptides 120-146WR (SEQ IDNO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26) comparedwith gentamicin as the comparator antibiotic. Each of the test peptideswas used at 25, 12.5 6.25, and 3.12 μM. The gentamicin was used at 4μg/ml. 1×10⁶/ml Pseudomonas aeruginosa ATCC® 27853™ (American TypeCulture Collection, Manassas, Va.) were incubated with the peptides,negative buffer control (tryptone saline), and positive controlgentamicin for 180 minutes at pH 7.2. 50 μl aliquots were plated out onagar plates and incubated overnight. Colony forming units were countedafter overnight incubation and CFU/ml calculated.

FIG. 13 shows bactericidal activity of CAP37 peptides 120-146WR (SEQ IDNO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26) comparedwith gentamicin as the comparator antibiotic. Each of the test peptideswas used at 25, 12.5, 6.25, and 3.12 μM. The gentamicin was used at 4μg/ml. 1×10⁶/ml Pseudomonas aeruginosa ATCC® 27853™ (American TypeCulture Collection, Manassas, Va.) were incubated with the peptides,negative buffer control (tryptone saline), and positive controlgentamicin for 180 minutes at pH 5.5. 50 μl aliquots were plated out onagar plates and incubated overnight. Colony forming units were countedafter overnight incubation and CFU/ml calculated.

FIG. 14 is a comparison of bactericidal activity of CAP37 peptides120-146WR (SEQ ID NO:30), 120-146WH (SEQ ID NO:31), 120-146QR (SEQ IDNO:28), 120-146QH (SEQ ID NO:29), 120-146QR-5RMP (SEQ ID NO:32), and120-146QH-5RMP (SEQ ID NO:33) for Pseudomonas aeruginosa ATCC® 27853™(American Type Culture Collection, Manassas, Va.). 1×10⁶/ml Pseudomonasaeruginosa ATCC® 27853™ were incubated with the peptides at 150, 75, and37.5 μg/ml and negative buffer control (tryptone saline) for 180 minutesat pH 5.5. 50 μl aliquots were plated out on agar plates and incubatedovernight. Colony forming units were counted after overnight incubationand CFU/ml calculated.

FIG. 15 shows that peptide 120-146QR-5RMP (SEQ ID NO:32) hasantimicrobial activity in a mouse model of Pseudomonas keratitis. Acircular wound was created on the mouse cornea by removing theepithelium, and the wound was infected with 10⁵ CFU of P. aeruginosa(ATCC® 27853™ (American Type Culture Collection, Manassas, Va.)).Infected wounds were treated with saline or saline containing indicatedconcentrations (2, 5, 10, and 20 mg/ml) of peptide every 30 minutes for6 hours on the first day. Infected wounds were treated twice on thesecond day and once on the third day. Mice were killed at 48 hourspost-infection, and the CFU/eye were quantified. The means are plottedand are representative of 5 mice per group. Mann Whitney test wasperformed for each group as compared to the saline control group.*P=0.015 and **P=0.097.

FIG. 16 shows that peptide 120-146WH (SEQ ID NO:31) has antimicrobialactivity in a mouse model of Pseudomonas keratitis. A circular wound wascreated on the mouse cornea by removing the epithelium, and the woundwas infected with 10⁵ CFU of P. aeruginosa (ATCC® 27853™ (American TypeCulture Collection, Manassas, Va.)). Infected wounds were treated withsaline or saline containing indicated concentrations (2, 5, 10, and 20mg/ml) of peptide every 30 minutes for 6 hours on the first day.Infected wounds were treated twice on the second day and once on thethird day. Mice were killed at 48 hours post-infection, and the CFU/eyewere quantified. The means are plotted and are representative of 4 to 5mice per group. Mann Whitney test was performed for each group ascompared to the saline control group.

FIG. 17 shows that peptide 120-146WR (SEQ ID NO:30) has antimicrobialactivity in a mouse model of Pseudomonas keratitis. A circular wound wascreated on the mouse cornea by removing the epithelium, and the woundwas infected with 10⁵ CFU of P. aeruginosa (ATCC® 27853™ (American TypeCulture Collection, Manassas, Va.)). Infected wounds were treated withsaline or saline containing indicated concentrations (2, 5, 10, and 20mg/ml) of peptide every 30 minutes for 6 hours on the first day.Infected wounds were treated twice on the second day and once on thethird day. Mice were killed at 48 hours post-infection, and the CFU/eyewere quantified. The means are plotted and are representative of 5 miceper group. ** P=0.0079 by Mann Whitney test as compared to the salinecontrol group.

FIG. 18 shows that CAP37 peptides 95-122 (SEQ ID NO:26), 120-146WH (SEQID NO:31), and 120-146QH-5RMP (SEQ ID NO:33) accelerate dermal woundhealing. Graphical results of in vivo wound healing in swine skin over14 days using saline as a control and three CAP37-based peptidecompounds used topically at 3 mg/ml are shown.

FIG. 19 contains representative results of dermal wound healing inswine, as evidenced by photographs of the results shown graphically inFIG. 18. Panel A: Saline vehicle control. Panel B: peptide 95-122 (SEQID NO:26). Panel C: peptide 120-146WH (SEQ ID NO:31). Panel D: peptide120-146QH-5RMP (SEQ ID NO:33).

FIG. 20 shows histopathology from swine dermal wound healing study basedon the criteria of inflammation, angiogenesis, granulation, andepithelialization of the results of FIGS. 18-19. The characteristicsthat were measured are required to indicate features of a well-healedwound. Inflammation should be low and re-epithelialization high.

FIG. 21 shows that CAP37 increases wound closure in HCEC monolayers. (A)HCECs were grown to confluency and scratched using a 10 μl pipette tip.Scratched HCEC monolayers were treated with HB-EGF (250 ng/ml) or rCAP37(25-2000 ng/ml), or were left untreated in basal Keratinocyte Serum FreeMedium (KSFM). Wound closure was monitored at 0, 18, 24, and 48 hoursutilizing a camera-equipped inverted microscope. The histogramrepresents data acquired at 48 hours, and the values are the mean±SEM ofthe percentage of wound closure. The data are representative of at least4 independent experiments. The percentage of wound closure in treatedmonolayers was compared to untreated controls by one-way analysis ofvariance (ANOVA) followed by Dunnett's multiple comparison test,**P<0.01, *P<0.05. (B) Representative images of scratched monolayerstreated with buffer control, HB-EGF, and rCAP37 (at 25, 100, and 250ng/ml) are shown for each time point. Images were taken at 20×objective.

FIG. 22 shows that CAP37 promotes corneal epithelial wound healing invivo. (A) The epithelium of the mouse cornea was removed using theAlgerBrush II, and corneal abrasions were treated at 0 and 16 hours withHB-EGF (250 ng/ml), rCAP37 (250 ng/ml), or vehicle control (normalsaline). Wound closure was monitored at 0, 16, 24, and 48 hours usingfluorescein staining and a camera-equipped inverted microscope. Data arerepresented as the percentage of wound closure and are expressed asmean±SEM. The data are representative of at least 6 mice per group. Thepercentage of wound closure in response to treatment was compared tovehicle-treated controls by unpaired t-test, **P<0.01, *P<0.05. (B)Representative images of mouse corneal wounds are shown at 0, 16, 24,and 48 hours following treatment with vehicle, HB-EGF, and CAP37. Blackdotted lines indicate the wound edge.

FIG. 23 is a histological analysis of corneal wound closure andre-epithelialization in response to CAP37. Corneas of mice were woundedusing the Algerbrush II and treated at 0 and 16 hours with rCAP37 (250ng/ml) or vehicle (normal saline). Whole eye globes were enucleated at0, 24, and 48 hours post wounding, and sections were stained usinghematoxylin and eosin (H&E). Representative images are shown of (A)CAP37-treated wound at 24 hours, (B) vehicle-treated wound at 24 hours,(C) CAP37-treated wound at 48 hours, and (D) vehicle treated wound at 48hours. The extent of closure and re-epithelialization of wounds wascompared with (E) normal unwounded cornea.

FIG. 24 shows that Protein Kinase C-delta (PKCδ) and Protein KinaseC-theta (PKCθ) are expressed in wounded and non-wounded HCEC monolayers.(A) HCECs (SV40 adenovirus immortalized cell line) were grown toconfluency and were scratched with a 10 μl pipette tip (right handpanel) or left unscratched (left hand panel). Monolayers were stainedfor PKC isoforms at 2 hours post wounding using anti-PKCδ antibody (250ng/ml), anti-PKCθ antibody (500 ng/ml), or IgG control (500 ng/ml), andanti-mouse secondary antibody (4 μg/ml, ALEXA FLUOR® 488 dye (LifeTechnologies Corp., Grand Island, N.Y.)). Representative images areshown. Scale bars, 20 μm. (B) Primary HCECs were stained to demonstrateconstitutive expression of PKC isoforms δ and θ in unwounded monolayers.Representative images are shown. Scale bars, 20 μm.

FIG. 25 shows that CAP37 treatment leads to an increase in PKCδ stainingin HCEC monolayers. HCEC monolayers were grown to confluency and werescratched with a 10 μl pipette tip or left unscratched, and then themonolayers were treated for 15 minutes with (A-B) buffer, (C-D) PMA (1μM), (E-F) 250 ng/ml CAP37, and (G-J) 500 ng/ml CAP37. Cells werestained for PKCδ (250 ng/ml; (A-H)), and PKCα (1 μg/ml; (I-J)) anddetected using immunofluorescence (4 μg/ml, ALEXA FLUOR® 488 dye (LifeTechnologies Corp., Grand Island, N.Y.)). Representative images (C-H)show staining for PKCδ in non-scratched monolayers (C, E, and G) andincreased staining in scratched monolayers (D, F, H) in response to PMAand CAP37. However, no increase in PKCα staining was observed followingtreatment of CAP37 (I-J). Scratched monolayers that were treated for 18hours with CAP37 (250 ng/ml; (L)) or left untreated (K) were stained forPKCδ. Treatment with CAP37 showed marked staining along the wound edgewas observed. Scale bars, 20 μm.

FIG. 26 shows that PKCδ is expressed along the leading edge of cornealepithelial wounds in vivo. Mice corneas were wounded using theAlgerBrush II and treated at 0 and 16 hours with vehicle (normal saline;(A-C)) or rCAP37 (250 ng/ml; (D-F)). Whole eye globes were enucleated at6 hours (A, B, D, E), 16 hours (C, F), and 48 hours (H) post-wounding,and sections were stained for PKCδ. Representative images showconstitutive staining for PKCδ in unwounded corneas (G). A lack ofstrong staining was seen at the leading wound edge (↓) in vehicletreated corneas at 6 hours (A, B) and 16 hours (C). Pronounced stainingfor PKCδ was seen along the leading wound edge (↓) in CAP37-treatedcorneas at 6 hours (D, E) and 16 hours (F). The presence of PKCδ wasstill seen 48 hours after wounding (H) and was comparable to theconstitutive staining seen at 0 hours in the unwounded cornea (G). Scalebars, 100 μm.

FIG. 27 shows that PKCδ is necessary for CAP37-induced wound healing invivo. (A) siRNA directed against PKCδ or scrambled siRNA was injectedinto the mouse conjunctiva. The mouse corneal epithelium was removedwith the AlgerBrush II. Corneal abrasions were treated at 0 and 16 hourswith rCAP37 (250 ng/ml), or were left untreated (saline vehiclecontrol). Wound healing was monitored at 0, 16, and 24 hours usingfluorescein staining. Data are represented as the percentage of woundclosure and are expressed as mean±SEM. The data are representative of atleast 9 mice per group. *P<0.05 by unpaired t-test as compared tovehicle (normal saline) treated controls. The efficiency of the PKCδknockdown in each cornea was confirmed by Western blot analysis 24 hourspost-injection and determined in comparison to the scrambledsiRNA-injected corneas. Data are representative of 22 experimentalsamples and are expressed as mean±SEM (***P<0.0005 by unpaired t-test).(B) Representative images stained with fluorescein indicating extent ofclosure of corneal abrasions at 0, 16, and 24 hours. The dotted line isused to demarcate the edge of the wound.

FIGS. 28A and 28B show the effect of BCC02-5R-MP (SEQ ID NO:21) incombination with Cefotaxime on MIC using Pseudomonas clinical isolateB64. Cefotaxime (Cefo) and peptide combinations included a constantamount of Cefotaxime (2.81 μg/ml) with suboptimal amounts of peptide (0,0.34, 1.01, and 3.04 μg/ml). FIG. 28A shows growth curve of Pseudomonasin the absence of Cefotaxime and antibiotic, Pseudomonas in the presenceof antibiotic (2.81 μg/ml), peptide (3.15 μg/ml), and antibiotic pluspeptide combinations. FIG. 28B is a histogram showing Fractional area(FA), which is an indication of the MIC. Combination of Cefotaxime with3.04 μg/ml of peptide shows significance. P=0.0143.

FIGS. 29A and 29B show the effect of BCC02-5R-MP (SEQ ID NO:21) incombination with Ciprofloxacin on MIC using Pseudomonas clinical isolateB64. Ciprofloxacin (Cipro) and peptide combinations included a constantamount of ciprofloxacin (2.1 μg/ml) with suboptimal amounts of peptide(0, 0.45, 1.35 and 4.05 μg/ml). FIG. 29A shows growth curve ofPseudomonas in the absence of Ciprofloxacin and antibiotic, Pseudomonasin the presence of antibiotic (2.1 μg/ml), peptide (4.05 μg/ml), andantibiotic plus peptide combinations. FIG. 29B is a histogram showingFractional area (FA), which is an indication of the MIC. Combination ofCiprofloxacin with 4.05 μg/ml of peptide shows significance. P=0.0003.

FIGS. 30A and 30B show the effect of BCC02-5R-MP (SEQ ID NO:21) incombination with Levofloxacin (Levo) and peptide combinations included aconstant amount of Levofloxacin (3.6 μg/ml) with suboptimal amounts ofpeptide (0, 0.34, 1.01, 3.04 μg/ml). FIG. 30A shows growth curve ofPseudomonas in the absence of Levofloxacin and antibiotic, Pseudomonasin the presence of antibiotic (3.6 μg/ml), peptide (3.04 μg/ml), andantibiotic plus peptide combinations. FIG. 30B is a histogram showingFractional area (FA), which is an indication of the MIC. Combination ofLevofloxacin with 3.04 μg/ml of peptide shows significance.

DETAILED DESCRIPTION

Before describing various embodiments of the presently disclosedinventive concepts in more detail by way of exemplary description,examples, and results, it is to be understood that the presentlydisclosed inventive concepts are not limited in application to thedetails of methods and compositions as set forth in the followingdescription. The presently disclosed inventive concepts are capable ofother embodiments or of being practiced or carried out in various ways.As such, the language used herein is intended to be given the broadestpossible scope and meaning; and the embodiments are meant to beexemplary, not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting unless otherwiseindicated as so. Moreover, in the following detailed description,numerous specific details are set forth in order to provide a morethorough understanding of the disclosure. However, it will be apparentto a person having ordinary skill in the art that the presentlydisclosed inventive concepts may be practiced without these specificdetails. In other instances, features which are well known to persons ofordinary skill in the art have not been described in detail to avoidunnecessary complication of the description.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concepts shall havethe meanings that are commonly understood by those having ordinary skillin the art. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which the presently disclosed inventiveconcepts pertain. All patents, published patent applications, andnon-patent publications referenced in any portion of this applicationare herein expressly incorporated by reference in their entirety to thesame extent as if each individual patent or publication was specificallyand individually indicated to be incorporated by reference.

All of the compositions and methods of production and applicationthereof disclosed herein can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions and methods of the presently disclosed inventive conceptshave been described in terms of particular embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit, and scope of the inventive concepts. All such similarsubstitutes and modifications apparent to those having ordinary skill inthe art are deemed to be within the spirit, scope, and concept of theinventive concepts as defined herein.

As utilized in accordance with the methods and compositions of thepresent disclosure, the following terms, unless otherwise indicated,shall be understood to have the following meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or when the alternatives are mutually exclusive,although the disclosure supports a definition that refers to onlyalternatives and “and/or.” The use of the term “at least one” will beunderstood to include one as well as any quantity more than one,including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 100, or any integer inclusive therein. The term “at least one”may extend up to 100 or 1000 or more, depending on the term to which itis attached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y and Z.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the composition, themethod used to administer the composition, or the variation that existsamong the study subjects.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, the term “substantially” means that thesubsequently described event or circumstance occurs at least 90% of thetime, or at least 95% of the time, or at least 98% of the time.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, aprotein that is present in a source that can be isolated from a sourcein nature and which has not been intentionally modified by man in thelaboratory or otherwise, other than to be purified, isnaturally-occurring.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as toxicity, irritationand/or allergic response commensurate with a reasonable benefit/riskratio.

By “biologically active” is meant the ability to modify thephysiological system of an organism without reference to how the activeagent has its physiological effects.

As used herein, “pure,” or “substantially pure” means an object species(e.g., a particular peptide compound) is the predominant species present(i.e., on a molar basis it is more abundant than any other active agentin the composition thereof), and particularly a substantially purifiedfraction is a composition wherein the object species comprises at leastabout 50 percent (on a molar basis) of all macromolecular speciespresent. Generally, a substantially pure composition will comprise morethan about 80% of all macromolecular species present in the composition,more particularly more than about 85%, more than about 90%, more thanabout 95%, or more than about 99%. Most particularly, the object speciesmay be purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.The term “pure” or “substantially pure” also refers to preparationswhere the object species (e.g., the peptide compound) is at least 60%(w/w) pure, or at least 70% (w/w) pure, or at least 75% (w/w) pure, orat least 80% (w/w) pure, or at least 85% (w/w) pure, or at least 90%(w/w) pure, or at least 92% (w/w) pure, or at least 95% (w/w) pure, orat least 96% (w/w) pure, or at least 97% (w/w) pure, or at least 98%(w/w) pure, or at least 99% (w/w) pure, or 100% (w/w) pure.

In certain compound names used herein, such as BCC01-5RMP, BCC02-5RMP,and BCC02-5RMP, “R” represents arginine, and “MP” represents asolubilizing moiety, such as a small PEG molecule (e.g., “AEEP”) asdefined elsewhere herein.

The terms “subject” and “patient” are used interchangeably herein andwill be understood to refer to a warm blooded animal, particularly amammal. Non-limiting examples of animals within the scope and meaning ofthis term include guinea pigs, dogs, cats, rats, mice, horses, goats,cattle, sheep, zoo animals, non-human primates, and humans.

“Treatment” refers to therapeutic treatments. “Prevention” refers toprophylactic or preventative treatment measures. The term “treating”refers to administering the composition to a patient for therapeuticpurposes.

The terms “therapeutic composition” and “pharmaceutical composition”refer to a peptide compound-containing composition that may beadministered to a subject by any method known in the art or otherwisecontemplated herein, wherein administration of the composition bringsabout a therapeutic effect as described elsewhere herein. Non-limitingexamples of modes of administration include oral, topical, retrobulbar,subconjunctival, transdermal, parenteral, subcutaneous, intranasal,intramuscular, intraperitoneal, intravitreal, and intravenous routes,including both local and systemic applications. In addition, thecompositions of the presently disclosed inventive concepts may bedesigned to provide delayed, controlled, extended, and/or sustainedrelease using formulation techniques which are well known in the art.

The term “topical” as used herein to define a mode of administration,means that a material is administered by being applied to the skin orinternally to an epithelial tissue.

The term “effective amount” refers to an amount of a peptide compoundwhich is sufficient to exhibit a detectable therapeutic effect withoutexcessive adverse side effects (such as toxicity, irritation andallergic response) commensurate with a reasonable benefit/risk ratiowhen used in the manner of the inventive concepts. The therapeuticeffect may include, for example but not by way of limitation, a partialor complete elimination of an infection or wound. The effective amountfor a patient will depend upon the type of patient, the patient's sizeand health, the nature and severity of the condition to be treated, themethod of administration, the duration of treatment, the nature ofconcurrent therapy (if any), the specific formulations employed, and thelike. Thus, it is not possible to specify an exact effective amount inadvance. However, the effective amount for a given situation can bedetermined by one of ordinary skill in the art using routineexperimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy,” and will beunderstood to mean that the subject in need of treatment is treated orgiven another drug for the condition (e.g., ibuprofen or a musclerelaxant) in conjunction with the pharmaceutical compositions of thepresently disclosed inventive concepts. This concurrent therapy can besequential therapy, where the patient is treated first with onecomposition and then the other composition, or the two compositions aregiven simultaneously. Non-limiting examples of combination therapies inaccordance with the presently disclosed inventive concepts include acombination of two or more of the peptides described herein, one or moreof said peptides in combination with one or more other antibiotics, orone or more of said peptides in combination with another drug given totreat a particular condition.

Abbreviations used herein, as well as the whole word/phrase representedthereby, are provided for reference hereinafter. AEEA(—NHCH₂CH₂OCH₂CH₂OCH₂CO— or NH₂CH₂CH₂OCH₂CH₂OCH₂CO—) is an N terminalsolubilizing group derived from [2-(2-amino-ethoxy)-ethoxy]-acetic acid(also known as 8-Amino-3,6-dioxaoctanoic acid). AEEEA(—NHCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO—) is anN terminal solubilizing group derived from{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-acetic acid (also known as11-Amino-3,6,9-trioxaundecanoic acid). AEEP (—NHCH₂CH₂OCH₂CH₂OCH₂CH₂CO—or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂CO—) is an N terminal solubilizing groupderived from [3-(2-amino-ethoxy)-ethoxy]-propanoic acid (also known as9-amino-4,7-dioxanonanoic acid). AEEEP(—NHCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CO— or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CO—)is an N terminal solubilizing group derived from[3-[2-(2-amino-ethoxy)-ethoxy]-ethoxy]-propanoic acid (also known as12-amino-4,7,10-trioxadodecananoic acid). ANOVA: analysis of variance.CAP37: cationic antimicrobial protein of molecular weight 37 KDa. BCA:bicinchoninic acid. BSA: bovine serum albumin. DAB: diaminobenzidinetetrahydrochloride. BPI: bactericidal permeability-increasing. DAG:diacyl glycerol. EDTA: ethylenediaminetetraacetic acid. EGF: epidermalgrowth factor. EGFR: epidermal growth factor receptor. ERK:extracellular-signal-regulated kinase. FBS: fetal bovine serum. GCSF:granulocyte colony-stimulating factor. GM-CSF: granulocyte macrophagecolony-stimulating factor. GPCR: G protein-coupled receptor. HB-EGF:heparin binding-epidermal growth factor. HBSS: Hank's balanced saltsolution. HCEC(s): human corneal epithelial cell(s). H&E: hematoxylinand eosin. HGF: hepatocyte growth factor. HBD-1: human beta-defensin-1.IL-6: interleukin-6. IL-8: interleukin-8. IP-10: interferon-inducibleprotein-10. KC: keratinocyte-derived chemokine. KSFM: keratinocyte serumfree media. LPS: lipopolysaccharide. MCP-1: monocyte chemotacticprotein-1. NADP: nicotinamide adenine dinucleotide phosphate. PBS:phosphate buffered saline. PDGF-BB: platelet derived growth factor-BB.PKC: protein kinase C. PMA: phorbol 12-myristate 13-acetate. PMSF:phenylmethylsulfonyl fluoride. RIPA: radioimmunoprecipitation assay.ROS: reactive oxygen species. SDS-PAGE: sodium dodecyl sulfatepolyacrylamide gel electrophoresis. SFM: serum free media. TBS:Tris-buffered saline. TBST: Tris-buffered saline TWEEN® 20 (ThermoFisher Scientific, Pittsburgh, Pa.). TGF-β: transforming growth factorbeta. TNF-α: tumor necrosis factor alpha.

Described herein in certain embodiments of the presently disclosedinventive concepts is a new generation of peptide compounds that haveenhanced antibacterial, antifungal, wound healing, and graft healingactivities and that can be scaled-up (i.e., have high solubility), andthat have high purity (in some embodiments at least 80%). In certainembodiments, modifications were made in the various peptides derivedfrom native CAP37 protein, including peptides derived from the 20-44(SEQ ID NO:7), 23-42 (SEQ ID NO:14), 95-122 (SEQ ID NO:26), 102-122 (SEQID NO:27), and 120-146 (SEQ ID NO:28) amino acid positions of CAP37protein. Various embodiments of the peptide compounds have shown highpurity, solubility, and potent activity against a number of bacterialspecies, including but not limited to, Pseudomonas aeruginosa (includingantibiotic resistant clinical isolates), Acinetobacter baumannii,Salmonella typhimurium, and Escherichia coli. In some embodiments, thepeptide compounds disclosed herein may be used to treat bacterial and/orfungal infections, surface wounds such as lacerations, abrasions,avulsions, incisions, and amputations, and “non-healing wounds” such asdiabetic ulcers (for example of the legs and feet), pressure sores, bedsores, wounds due to peripheral vascular disease, “non-healing” or“slow-healing” post-surgical wounds, and burns. The peptide compounds incertain embodiments may be used as treatments to enhance acceptance ofgrafts such as skin grafts and organ grafts. The peptide compounds incertain embodiments may be used as treatments for ocular infections,ocular wounds and abrasions, and ocular ulcers and treatment ofinflammatory conditions such as dry eye. The peptides may be used incombination with each other, in combination with other antibiotics, orin combination with another drug given to treat a particular condition.

Certain embodiments of the presently disclosed inventive concepts aredirected to a composition that comprises a peptide compound representedby Formula (I) below:S—X_(R)-Pep-Y_(R)wherein Pep is one of SEQ ID NOs:1-14, 25-31, and 47 or anotherappropriate amino acid sequence described herein; X_(R) and Y_(R) areeach independently 0, 1, 2, 3, 4, 5, or 6 arginine residues, with theproviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; and S is asolubilizing moiety made up of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (i.e.,1-10) small PEG subunits having the formulaNH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CH₂)_(q)CO— wherein q=0, 1, 2, 3, or 4 andp=1, 2, 3, 4, 5, 6, 7, or 8, and n=1 or 2 wherein n=2 if the subunit isterminal. For example, in certain non-limiting embodiments, S maycomprise one subunit AEEP (NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂CO—), or two subunitsAEEP (NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂CONHCH₂CH₂OCH₂CH₂OCH₂CH₂CO—), or onesubunit AEEEP (NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CO—), or two subunits AEEEP(NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CONHCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CO—), orone AEEP and one AEEEP in any order. The 1-10 small PEG subunits may beidentical or may be a combination of different small PEG subunits (inany order) having the formula —NHCH₂CH₂—(OCH₂CH₂)_(p)—O(CH₂)_(q)CO— asdescribed above. In at least one embodiment, S comprises at least onesubunit which is not AEEA (q=1, p=1), or AEEEA (q=1, p=2). For example,in one embodiment S comprises one of (i) AEEA and AEEP, (ii) AEEA andAEEEP, (iii) AEEEA and AEEP, or (iv) AEEEA and AEEEP, but does notinclude (i) AEEA and AEEA, (ii) AEEA and AEEEA, or (iii) AEEEA andAEEEA.

The —O(CH₂)_(q)CO— portion (“head group”) of the PEG subunit (S) whichis attached to the peptide (X_(R)-Pep-Y_(R)) may be further modified tocomprise a number of alternate embodiments of the (CH₂)_(q) portion ofthe head group. These alternate embodiments, as well as those describedabove, can be characterized by Formula (II) below:—O(CR₁R₂)_(q)CO—wherein R₁ and R₂ of Formula II are side groups on C (i.e., “R₁—C—R₂”)wherein q=0-4, and R₁ and R₂ are selected from H, CH₃, and CH₂CH₃. Whenq=1, either or both of R₁ and R₂ can be selected from H, CH₃, andCH₂CH₃. When q=2, either of the two R₁ groups and either of the two R₂groups can be selected from H, CH₃, and CH₂CH₃. When q=3, any of thethree R₁ groups and any of the three R₂ groups can be selected from H,CH₃, and CH₂CH₃. When q=4, any of the four R₁ groups and any of the fourR₂ groups can be selected from H, CH₃, and CH₂CH₃. Thus, in thesenon-limiting embodiments, the head group may comprise one or moresubstitutions for H on the carbons of the carbon backbone when C=1-4.

Examples of embodiments of head groups having Formula II include, butare not limited to:

-   -   1. —OCH₂CH₂CO— (e.g., as in AEEP).    -   2. —OCH₂CO— (e.g., as in AEEA).    -   3. —OCH₂CH₂CH₂CO— (wherein q=3).    -   4. —OCH₂CH₂CH₂CH₂CO— (wherein q=4 i.e., a pentanoic head group).    -   5. —OC(CH₃)₂CO— (wherein R₁ and R₂=CH₃ and q=1).    -   6. —OCH₂C(CH₃)₂CO— (wherein R₁ and R₂=H on the first carbon and        R₁ and R₂=CH₃ on the second carbon and q=2).    -   7. —OCH₂CH₂CH₂CH(CH₃)CO— (wherein R₁=H and R₂=CH₃ on one carbon,        and R₁ and R₂=H on every other carbon and q=4).    -   8. —OCH₂CH₂CH₂C(CH₃)₂CO— (wherein R₁ and R₂=CH₃ on one carbon,        and R₁ and R₂=H on all other carbons).    -   9. —OCH₂CH₂CH₂CH(CH₂CH₃)CO— (wherein R₁=H and R₂=CH₂CH₃ on one        carbon, and R₁ and R₂=H on all other carbons).    -   10. —OCH(CH₃)CH₂CH₂CH(CH₂CH₃)CO— (wherein R₁=H and R₂=CH₃ on one        carbon, R₁=H and R₂=CH₂CH₃ on one carbon, and R₁ and R₂=H on all        other carbons).        All stereoisomers of the peptide compounds formed based on the        solubilizing units described for Formula II are intended to be        included in the presently disclosed inventive concepts.

In one embodiment, X_(R) includes four arginine residues, while Y_(R) isone arginine residue. In addition, the S group may include, as indicatedabove, two AEEP moieties for example. Particular non-limiting examplesof peptide compounds in accordance with the presently disclosedinventive concepts include one or more peptide compounds that have thesequence of one of SEQ ID NOs:21-23 and 32-46, as described in furtherdetail herein below.

In one non-limiting embodiment, the “Pep” sequence of the Formula (I)peptide compound described herein may comprise the following sequence(SEQ ID NO:25):R—H—X₃—X₄—X₅—X₆—X₇—X₈—X₉—H—X₁₁—R—X₁₃—X₁₄-M-X₁₆—X₁₇—X₁₈—X₁₉—X₂₀wherein X₃ and X₁₃ are phenylalanine, tyrosine, arginine, lysine orhistidine; X₄ is selected from cysteine, serine, threonine, andmethionine; X₅ and X₆ are selected from glycine and alanine; X₇, X₁₁,and X₁₄ are selected from alanine, leucine, isoleucine, and valine; X₉,X₁₇, and X₁₈ are selected from alanine, leucine, isoleucine, and valine;X₁₆ is selected from serine, threonine, and methionine; X₁₉ is selectedfrom serine, threonine and methionine; X₂₀ is selected from cysteine,serine, and methionine; R is arginine; H is histidine; and M ismethionine. This sequence is a derivative of SEQ ID NO:8 (i.e., aminoacids 23-42 of CAP37 protein).

In another non-limiting embodiment, the “Pep” sequence of the Formula(I) peptide compound described herein may comprise the followingsequence (SEQ ID NO:47):R—H—X₃—X₄—X₅—X₆—X₇—X₈—X₉—H—X₁₁—R—X₁₃—X₁₄-M-X₁₆—X₁₇—X₁₈—X₁₉—X₂₀wherein X₃ and X₁₃ are phenylalanine, tyrosine, arginine, lysine, orhistidine; X₄ is selected from cysteine, serine, threonine, andmethionine; X₅ and X₆ are selected from glycine and alanine; X₇, X₁₁,and X₁₄ are selected from alanine, leucine, isoleucine, and valine; X₉,X₁₇, and X₁₈ are selected from alanine, leucine, isoleucine, and valine;X₁₆ is selected from serine, threonine, and methionine; X₁₉ is selectedfrom serine, threonine, and methionine; X₂₀ is selected from cysteine,serine, threonine, and methionine; R is arginine; H is histidine; and Mis methionine.

Certain embodiments of the presently disclosed inventive conceptsinclude peptide compounds having the sequences of at least one of SEQ IDNOs:8-14 (i.e., sequences similar to SEQ ID NOs: 1-7, respectively,wherein the N-terminal three amino acids (NQG) and the C-terminal twoamino acids (FQ) are truncated such that the Pep portion of the peptidecompound contains 20 amino acids rather than 25 amino acids). In otherembodiments of the presently disclosed inventive concepts, one or moreof the following substitutions may be made in one or more of SEQ IDNOs:7 and 14: phenylalanine replaced by tyrosine; glycine replaced byalanine; valine replaced by alanine, leucine, or isoleucine; alaninereplaced by leucine, isoleucine, or valine; leucine replaced by alanine,isoleucine, or valine; isoleucine replaced by valine, leucine, oralanine; serine replaced by threonine or methionine, and threoninereplaced by serine or methionine; and the cysteine residues may besubstituted with a serine, threonine, or methionine, as long as onecysteine of the two cysteine residues remains present. Similarly, one ormore conservative substitutions may be made in one or more of SEQ IDNOs: 26-35 when these sequences are used in the peptide compoundsdescribed herein.

One non-limiting example of a composition constructed in accordance withthe presently disclosed inventive concepts includes a compositioncomprising the peptide of SEQ ID NO:21[(AEEP)-(AEEP)-RRRRNQGRHFSGGALIHARFVMTAASCFQR], and wherein the peptidecompound is effective in enhancing the therapeutic efficacy of anantibiotic in a subject when administered to the subject in combinationwith the antibiotic. Another non-limiting example of a compositionconstructed in accordance with the presently disclosed inventiveconcepts includes a composition comprising the peptide of SEQ ID NO:32[(AEEP)-(AEEP)-RRRRGTRCQVAGWGSQRSGGRLSRFPRFVNVR], and wherein thepeptide compound has wound healing and antibacterial activity in asubject in need of such therapy.

The compositions of the presently disclosed inventive concepts maycontain multiple peptide compounds, wherein each peptide compound isrepresented by Formula (I) as described herein above. For example butnot by way of limitation, the composition may include at least twopeptide compounds, wherein each of the at least two peptide compounds isfurther defined as having the sequence of one of SEQ ID NOs:21-23 and32-46. In another non-limiting example, the composition may include atleast two peptide compounds as represented by Formula (I), wherein Pepof a first peptide compound is one of SEQ ID NOs:1-14 and 25, and Pep ofa second peptide compound is one of SEQ ID NOs:26-31. In yet anothernon-limiting example, the composition may include at least two peptidecompounds as represented by Formula (I), wherein Pep of a first peptidecompound is one of SEQ ID NOs:26-27, and Pep of a second peptidecompound is one of SEQ ID NOs:28-31.

Further, when multiple peptide compounds are present in the compositionsof the presently disclosed inventive concepts, the multiple peptidecompounds may be multimerized to form homomultimers or heteromultimersby linkage across the cysteine residues. One non-limiting example ofmultimerization that may be utilized in accordance with the presentlydisclosed inventive concepts is dimerization to form homodimers and/orheterodimers. One non-limiting method of multimerization includeslinkage via “intermolecular oxidation,” in which the thiol groupattached to the cysteine at position 26 could be linked to a SH groupfrom another Cys 26 peptide, thus giving a homodimer. In anothernon-limiting dimerization method, the SH group at Cys 42 could becyclized with the SH group on Cys 42 from another peptide, giving a Cys42 homodimer. A third non-limiting alternative includes the linkage ofthe thiol groups between Cys 42 and Cys 26 of separate peptides to givea heterodimer.

The compositions of the presently disclosed inventive concepts may alsocontain at least one additional therapeutically active agent. Anytherapeutically active agent known in the art or otherwise contemplatedherein may be combined with the peptide compound in the compositions ofthe presently disclosed inventive concepts. Non-limiting examples oftherapeutically active agents that may be utilized in accordance withthe presently disclosed inventive concepts include antibiotics, othertherapeutically active peptides, antibacterial agents, antifungalagents, wound healing agents, and/or graft acceptance agents.

Any therapeutically active peptide known in the art or otherwisecontemplated herein may be combined with the peptide compounds in thecomposition of the presently disclosed inventive concepts. Non-limitingexamples of therapeutically active peptides include CAP37 peptides, suchas but not limited to, the peptides of SEQ ID NOs:26-28. One particularnon-limiting example of a composition contains (a) at least one peptidecompound of one of SEQ ID NOs:21-23, and (b) at least one additionaltherapeutic peptide of one of SEQ ID NOS:26-28. Another particularnon-limiting example of a composition contains (a) at least one peptidecompound of one of SEQ ID NOs:32-35, and (b) at least one additionaltherapeutic peptide of one of SEQ ID NOS:26-27.

Any antibiotic known in the art or otherwise contemplated herein may beutilized in accordance with the presently disclosed inventive concepts.Examples of antibiotics (e.g., antibacterial agents) which may be usedin combination with the peptide compounds of the present disclosureinclude, but are not limited to, gentamicin, amikacin, kanamycin,tobramycin, neomycin, ertapenem, doripenem, imipenem/cilastatin,meropenem, ceftazidime, cefepime, ceftaroline, ceftobiprole, aztreonam,piperacillin, polymyxin B, Colistin, ciprofloxacin, levofloxacin,moxifloxacin, gatifloxacin, tigecycline, and combinations andderivatives thereof. In particular, the following non-limiting examplesof antibiotics may be used in combination with peptide compounds thatpromote wound healing: gentamicin, amikacin, kanamycin, tobramycin,neomycin, ertapenem, doripenem, imipenem/cilastatin, meropenem,ceftazidime, cefepime, ceftaroline, ceftobiprole, aztreonam,piperacillin, polymyxin B, Colistin, ciprofloxacin, levofloxacin,moxifloxacin, gatifloxacin, tigecycline, clindamycin, clarithromycin,vancomycin, azithromycin, cefixime, ceftriaxone, cefamandole,cefotaxime, cefdinir, bacitracin, sulfacetamide, doxycycline, andcombinations and derivatives thereof.

The peptide compounds of the compositions of the presently disclosedinventive concepts may be produced by any methods known in the art. Forexample, the peptide compounds may be produced synthetically or may beproduced by recombinant methods. Thus, other embodiments of thepresently disclosed inventive concepts include a DNA molecule having anucleotide sequence that encodes a portion or all of the compositionsdescribed herein above. For example, the DNA molecule may have anucleotide sequence encoding a peptide compound having an amino acidsequence as defined in any of the amino acid sequences listed,described, or otherwise contemplated herein, including for example butnot by way of limitation, those having substituted cysteine residues atpositions 26 or 42.

Other embodiments of the presently disclosed inventive concepts includepharmaceutical compositions that contain a therapeutically-effective orpharmaceutically-effective amount of at least one active ingredient(i.e., one or more of the compositions described herein above) incombination with a pharmaceutically-acceptable carrier. As used herein,a “pharmaceutically acceptable carrier” is a pharmaceutically acceptablesolvent, suspending agent, or vehicle for delivering the compositions ofthe presently disclosed inventive concepts to the subject. The carriermay be, for example but not by way of limitation, liquid or solid, andthe carrier may be selected with the planned manner of administration inmind. Examples of pharmaceutically acceptable carriers that may beutilized in accordance with the presently disclosed inventive conceptsinclude, but are not limited to, polyethylene glycol (PEG), polymers,liposomes, ethanol, DMSO, aqueous buffers, solvents, oils, DPPC, lipids,and combinations thereof.

Particular non-limiting examples of pharmaceutical compositionsconstructed in accordance with the presently disclosed inventiveconcepts include: (a) a pharmaceutical composition comprising acomposition that includes a peptide compound as represented by Formula(I) in combination with a pharmaceutically-acceptable carrier; (b) apharmaceutical composition comprising at least two peptide compounds asrepresented by Formula (I) in combination with a pharmaceutically-activecarrier; and (c) a pharmaceutical composition comprising at least onecomposition that includes a peptide compound as represented by Formula(I) in combination with at least one therapeutically active agent (suchas but not limited to, a peptide compound or an antibiotic) and apharmaceutically-acceptable carrier.

The pharmaceutical compositions may contain, in addition to the peptidecompound and pharmaceutically-acceptable carrier, one or more additionalcomponents, including but not limited to, diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. Suitable carriers, vehicles, and other components that may beincluded in the formulation are described, for example, in Remington:The Science and Practice of Pharmacy, 21^(st) ed. The term“pharmaceutically acceptable” means a non-toxic material that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). The characteristics of the carrier will depend onthe route of administration.

The pharmaceutical composition of the presently disclosed inventiveconcepts may be in the form of a liposome in which the peptide compoundis disposed, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 4,737,323, all of which are incorporatedherein by reference.

As is evident from the above, in various embodiments, the peptidecompounds of the presently disclosed inventive concepts (e.g., ascharacterized by Formula (I)) find uses as antibacterial and/orantifungal therapeutics, as treatments for ocular infections, ocularwounds or abrasions and/or ocular ulcer treatments, ocular dry eyetreatments, as surface wound treatments to promote healing, and/or astreatments to promote healing and acceptance of skin and/or organgrafts.

Thus, certain embodiments of the presently disclosed inventive conceptsinclude a method of treating and/or inhibiting a bacterial and/or fungalinfection in a patient, subject, and/or mammal, and/or a method ofprophylactically preventing (and/or reducing the occurrence of) abacterial and/or fungal infection in a patient, subject, and/or mammal;in this method, any of the compositions described herein above orotherwise contemplated herein is administered to the patient, subject,and/or mammal. In particular non-limiting embodiments, the bacterialinfection is caused by a Gram negative bacterium, such as but notlimited to, Pseudomonas aeruginosa, Escherichia coli, Salmonellatyphimurium, and Acinetobacter baumannii. In other non-limitingembodiments, the fungal infection is caused by a fungal organismselected from the group consisting of Candida spp., Saccharomycescerevisiae, Histoplasma spp., Histoplasma capsulatum, Aspergillus spp.,Aspergillus fumigatus, and Cryptococcus neoformans.

In certain embodiments, the presently disclosed inventive concepts alsoinclude methods (including but not limited to, topical and/or systemicmethods) of treating ocular wounds, ocular infections, ulcers, and/orinfections in a patient, subject, and/or mammal. In additionalembodiments, the presently disclosed inventive concepts also includemethods of treating surface wounds in a patient to promote healing ofthe wound; in this method, any of the compositions described hereinabove or otherwise contemplated herein is administered (such as but notlimited to, topically and/or systemically) to the patient, subject,and/or mammal. Non-limiting examples of such wounds which can be treatedwith the peptide compounds described herein include lacerations,abrasions, avulsions, incisions, and amputations, and “non-healingwounds” such as diabetic ulcers (for example of the legs and feet),pressure sores, wounds due to peripheral vascular disease, burns,infected wounds, and surgical wounds.

In other embodiments, the presently disclosed inventive concepts alsoinclude methods of treating organ graft(s) and/or skin graft(s) forpromoting the healing and acceptance thereof in a patient, subject,and/or mammal; in this method, any of the compositions described hereinabove or otherwise contemplated herein is administered (such as but notlimited to, topically and/or systemically) to the patient, subject,and/or mammal.

The peptide compounds described or otherwise contemplated herein can beadministered (a) in combination with one another, and/or (b) incombination with at least one additional therapeutic agent (such as butnot limited to, an antibacterial agent (antibiotic), an anti-fungalagent, or other therapeutically active peptides, including but notlimited to, other CAP37 peptides). Examples of such antibacterial agentsare described above. Examples of antifungal agents which can beadministered in combination with the presently described peptidecompounds include, but are not limited to, those in the followingclasses: polyenes (e.g., Nystatin, Amphotericin B, and Pimaricin),azoles (e.g., Ketoconazole, Fluconazole, Itraconazole, Voriconazole, andPosaconazole), allylamine and morpholines (e.g., Naftifine, Terbinafine,and amorolfine), echinocandins (e.g. Micafungin, Caspofungin, andPneumocandin), and anti-metabolites (e.g., 5-Fluorocytosine).

The phrase “in combination” as used in this context means that thepeptide compound(s) and/or the therapeutic agent(s) are givensubstantially contemporaneously, simultaneously, and/or wholly orpartially sequentially; when delivered wholly or partially sequentially,the agent may be administered before and/or after the peptide compound,and vice versa. In a non-limiting example, the compound is used in acombination therapy with conventional antibiotic chemotherapeuticsand/or treatments, for example antibiotics to which bacterial organismshave generally developed a resistance.

Certain other embodiments of the presently disclosed inventive conceptsinclude methods of enhancing the efficacy of an antibiotic in thetreatment of a bacterial infection. In one embodiment of the method, atherapeutically-effective amount of any of the compositions described orotherwise contemplated herein that comprises an antibiotic and a peptidecompound is administered to the subject. The therapeutically-effectiveamount of the composition includes an amount of the antibiotic that: (i)has suboptimal activity or is ineffective against the bacteria whenadministered alone, and (ii) is effective against the bacteria whenadministered in combination with the peptide compound.

Another embodiment of the method involves administration to the subjectof two separate components: (a) a composition as described or otherwisecontemplated herein and that comprises a peptide compound, and (b) anantibiotic. The antibiotic is administered in an amount that: (i) hassuboptimal activity or is ineffective against the bacteria whenadministered alone, and (ii) is effective against the bacteria whenadministered in combination with the peptide compound. The antibioticand composition may be administered simultaneously or wholly orpartially sequentially; when administered wholly or partiallysequentially, either component may be administered first.

Certain embodiments of the presently disclosed inventive conceptsinclude methods of enhancing the efficacy of an antibiotic in thetreatment of at least one of a wound and a graft to promote healing ofthe wound and/or acceptance of the graft in a subject in need of suchtreatment. In one embodiment of the method, a therapeutically-effectiveamount of any of the compositions described or otherwise contemplatedherein that comprises an antibiotic and a peptide compound isadministered to the subject. The therapeutically-effective amount of thecomposition includes an amount of the antibiotic that: (i) hassuboptimal activity or is ineffective in promoting healing of the woundand/or acceptance of the graft when administered alone, and (ii) iseffective in promoting healing of the wound and/or acceptance of thegraft when administered in combination with the peptide compound.

Another embodiment of the method involves administration to the subjectof two separate components: (a) a composition as described or otherwisecontemplated herein and that comprises a peptide compound, and (b) anantibiotic. The antibiotic is administered in an amount that: (i) hassuboptimal activity or is ineffective in promoting healing of the woundand/or acceptance of the graft when administered alone, and (ii) iseffective in promoting healing of the wound and/or acceptance of thegraft when administered in combination with the peptide compound. Theantibiotic and composition may be administered simultaneously or whollyor partially sequentially; when administered wholly or partiallysequentially, either component may be administered first.

A therapeutically effective amount of a peptide compound of thepresently disclosed inventive concepts refers to an amount which iseffective in controlling, reducing, and/or inhibiting a bacterial and/orfungal infection. The term “controlling” is intended to refer to allprocesses wherein there may be a slowing, interrupting, arresting,and/or stopping of the progression of the infection and does notnecessarily indicate a total elimination of the infection symptoms.

The term “therapeutically effective amount” is further meant to definean amount resulting in the improvement of any parameters or clinicalsymptoms characteristic of a bacterial and/or fungal infection. Theactual dose will vary with the patient's overall condition, theseriousness of the symptoms, and counter indications. As used herein,the term “therapeutically effective amount” also means the total amountof each active component of the pharmaceutical composition or methodthat is sufficient to show a meaningful patient benefit, e.g., areduction of bacterial and/or fungal infection and/or an improvement inwound and/or graft healing. When applied to an individual activeingredient that is administered alone, the term refers to thatingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially,and/or simultaneously.

A therapeutically effective amount of the peptide compound used in thetreatment described herein can be determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining the therapeutically effective dose, anumber of factors may be considered by the attending diagnostician,including, but not limited to: the species of the subject; its size,age, and general health; the specific bacterial or fungal disease orother condition involved; the degree of or involvement or the severityof the bacterial or fungal disease or other condition; the response ofthe individual subject; the particular compound administered; the modeof administration; the bioavailability characteristic of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances. A therapeuticallyeffective amount of a peptide compound of the presently disclosedinventive concepts also refers to an amount of the peptide compoundwhich is effective in controlling or reducing the bacterial or fungalinfection, or improving wound or graft healing, for example.

A therapeutically effective amount of a composition of the presentlydisclosed inventive concepts will generally contain sufficient activeingredient (i.e., the peptide compound) to deliver from about 0.1 μg/kgto about 100 mg/kg (weight of active ingredient/body weight of patient).Particularly, the composition will deliver about 0.5 μg/kg to about 50mg/kg, and more particularly about 1 μg/kg to about 10 mg/kg.

Practice of the method of the presently disclosed inventive concepts maycomprise administering to a subject a therapeutically effective amountof the peptide compound in any suitable systemic and/or localformulation, in an amount effective to deliver the dosages listed above.An effective, particular dosage of the peptide compound forsubstantially inhibiting the bacterial and/or fungal infection is about1 μg/kg to about 10 mg/kg of the peptide. The dosage can beadministered, for example but not by way of limitation, on a one-timebasis, or administered at multiple times (for example but not by way oflimitation, from one to five times per day, or once or twice per week),or continuously via a venous drip, depending on the desired therapeuticeffect. In one non-limiting example of a therapeutic method of thepresently disclosed inventive concepts, the peptide compound is providedin an IV infusion in the range of from about 1 mg/kg to about 10 mg/kgof body weight once a day.

In practicing the method of treatment or use of the presently disclosedinventive concepts, a therapeutically effective amount of the peptidecompound is administered to a mammal having a bacterial and/or fungaldisease state, and/or other condition desired to be treated (such as butnot limited to, a wound or a graft). The peptide compound may beadministered in accordance with the method of the presently disclosedinventive concepts either alone or in combination with other therapies.

Administration of the peptide compound used in the pharmaceuticalcomposition or to practice the method of the presently disclosedinventive concepts can be carried out in a variety of conventional ways,such as, but not limited to, orally, by inhalation (e.g., for sinusfungal infections), rectally, or by cutaneous, subcutaneous,intraperitoneal, vaginal, or intravenous injection. Oral formulationsmay be formulated such that the peptide compound passes through aportion of the digestive system before being released, for example itmay not be released until reaching the small intestine, or the colon.

When a therapeutically effective amount of the peptide compound isadministered orally, the compound may be in the form of a tablet,capsule, powder, solution, or elixir. The pharmaceutical composition mayadditionally contain a solid carrier, such as a gelatin or an adjuvant.The tablet, capsule, and powder particularly contains from about 0.05 toabout 95% of the peptide compound by dry weight. When administered inliquid form, a liquid carrier such as water, petroleum, oils of animalor plant origin such as peanut oil, mineral oil, soybean oil, or sesameoil, or synthetic oils may be added. The liquid form of thepharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol, or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition particularlycontains from about 0.005 to about 95% by weight of peptide. Forexample, a dose of about 10 mg to about 1000 mg once or twice a daycould be administered orally.

For oral administration, the peptide compounds can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions, or emulsions. Solid unit dosage forms canbe capsules of the ordinary gelatin type containing, for example,surfactants, lubricants, and inert fillers such as lactose, sucrose, andcornstarch, or the dosage forms can be sustained release preparations.

In another embodiment, the peptide compounds of the presently disclosedinventive concepts can be tableted with conventional tablet bases suchas lactose, sucrose, and cornstarch in combination with binders, such asacacia, cornstarch, or gelatin, disintegrating agents such as potatostarch or alginic acid, and a lubricant such as stearic acid ormagnesium stearate. Liquid preparations are prepared by dissolving thepeptide compound in an aqueous or non-aqueous pharmaceuticallyacceptable solvent which may also contain suspending agents, sweeteningagents, flavoring agents, and preservative agents as are known in theart.

For parenteral administration, for example, the peptide compounds may bedissolved in a physiologically acceptable pharmaceutical carrier andadministered as either a solution or a suspension. Non-limiting examplesof suitable pharmaceutical carriers are water, saline, dextrosesolutions, fructose solutions, ethanol, or oils of animal, vegetative,or synthetic origin. The pharmaceutical carrier may also containpreservatives and buffers as are known in the art.

When a therapeutically effective amount of the peptide compound isadministered by intravenous, cutaneous, or subcutaneous injection, thepeptide compound is particularly in the form of a pyrogen-free,parenterally acceptable aqueous solution or suspension. The preparationof such parenterally acceptable peptide solutions, having due regard topH, isotonicity, stability, and the like, is well within the skill inthe art. A particular pharmaceutical composition for intravenous,cutaneous, or subcutaneous injection may contain, in addition to thepeptide compound, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle as known in theart. The pharmaceutical composition of the presently disclosed inventiveconcepts may also contain stabilizers, preservatives, buffers,antioxidants, or other additive(s) known to those of skill in the art.

As noted above, the compositions can also include an appropriatecarrier. For topical use, any of the conventional excipients may beadded to formulate the peptide compound into a lotion, ointment, powder,cream, spray, or aerosol. For surgical implantation, the peptidecompound may be combined with any of the well-known biodegradable andbioerodible carriers, such as polylactic acid and collagen formulations.Such materials may be in the form of solid implants, sutures, sponges,wound dressings, and the like. In any event, for local use of thematerials, the peptide compound is usually present in the carrier orexcipient in a weight ratio of from about 1:1000 to about 1:20,000, butis not limited to ratios within this range. Preparation of compositionsfor local use is detailed in Remington: The Science and Practice ofPharmacy, 21^(st) ed.

As noted, particular amounts and modes of administration can bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration, depending upon the particular characteristics of thepeptide compound selected, the infection to be treated, the stage of theinfection, and other relevant circumstances using formulation technologyknown in the art, described, for example, in Remington: The Science andPractice of Pharmacy, 21^(st) ed.

The pharmaceutical compositions of the presently disclosed inventiveconcepts can be manufactured utilizing techniques known in the art.Typically, the therapeutically effective amount of the peptide compoundwill be admixed with a pharmaceutically acceptable carrier.

The presently disclosed inventive concepts further includes (but is notlimited to) a method of treating a topical bacterial or fungal infectionby topically applying an amount of the composition sufficient to treatthe infection, e.g., about 0.5% to about 10% by weight of thecomposition. The topical medication may take any number of standardforms such as pastes, gels, creams, and ointments. In one embodiment, asolution of the composition to be administered may be prepared using asolvent known to promote transdermal absorption, such as but not limitedto, ethanol or dimethyl sulfoxide (DMSO) with or without otherexcipients. Particularly, topical administration may be accomplishedusing a patch either of the reservoir and porous membrane type or of asolid matrix variety.

The amount of the peptide compound in the pharmaceutical composition ofthe presently disclosed inventive concepts will depend upon the natureand severity of the condition being treated and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of peptide compound with which to treateach individual patient. Initially, the attending physician mayadminister low doses of the peptide compound and observe the patient'sresponse. Larger doses may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. Without wishing to be held to a specific dosage, itis contemplated that the various pharmaceutical compositions used topractice the method of the presently disclosed inventive concepts maycontain, but are not limited to, about 0.1 mg to about 100 mg of thepeptide compound per kg body weight per dose.

The duration of intravenous therapy using the pharmaceutical compositionof the presently disclosed inventive concepts will vary, depending onthe severity of the disease being treated and the condition andpotential idiosyncratic response of each individual patient. It iscontemplated that the duration of each application of the peptidecompound may be in the range of about 1 to about 2 hours and given onceabout every 12 or 24 hours by continuous intravenous administration.Other antibiotics, intravenous fluids, and cardiovascular andrespiratory support could also be provided if requested by the attendingphysician in a manner known to one of ordinary skill in the art.

Bacteria which may be treated by the peptide compounds of the presentlydisclosed inventive concepts include, but are not limited to, Gramnegative bacteria such as but not limited to Pseudomonas aeruginosa,Escherichia coli, Salmonella typhimurium and Acinetobacter baumannii.Fungal infections which may be treated by the peptide compoundsdescribed herein include but are not limited to those caused by Candidaspp., Saccharomyces cerevisiae, Histoplasma capsulatum and otherHistoplasma species which cause histoplasmosis, Aspergillus fumigatusand other species (occurring mostly in the lung) which causesAspergillosis, and Cryptococcus neoformans (sometimes found in the lungbut mostly in the CNS), which causes a disease known as cryptococcosis.

Additional pharmaceutical methods may be employed to control theduration of action of the peptide compound. Increased half-life and/orcontrolled release preparations may be achieved through the use ofpolymers to conjugate, complex with, and/or absorb the peptide describedherein. The controlled delivery and/or increased half-life may beachieved by selecting appropriate macromolecules (for example but not byway of limitation, polysaccharides, polyesters, polyamino acids,homopolymers polyvinyl pyrrolidone, ethylenevinylacetate,methylcellulose, or carboxymethylcellulose, and acrylamides such asN-(2-hydroxypropyl) methacrylamide), and the appropriate concentrationof macromolecules as well as the methods of incorporation, in order tocontrol release.

Another possible method useful in controlling the duration of action bycontrolled release preparations and half-life is incorporation of thepeptide compound or its functional derivatives into particles of apolymeric material such as polyesters, polyamides, polyamino acids,hydrogels, poly(lactic acid), ethylene vinylacetate copolymers,copolymer micelles of, for example, PEG and poly(I-aspartamide).

It is also possible to entrap the peptide compounds in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are well known topersons having ordinary skill in the art.

When the peptide composition is to be used as an injectable material, itcan be formulated into a conventional injectable carrier. Suitablecarriers include biocompatible and pharmaceutically acceptable phosphatebuffered saline solutions, which are particularly isotonic.

For reconstitution of a lyophilized product in accordance with thepresently disclosed inventive concepts, one may employ a sterilediluent, which may contain materials generally recognized forapproximating physiological conditions and/or as required bygovernmental regulation. In this respect, the sterile diluent maycontain a buffering agent to obtain a physiologically acceptable pH,such as sodium chloride, saline, phosphate-buffered saline, and/or othersubstances which are physiologically acceptable and/or safe for use. Ingeneral, the material for intravenous injection in humans should conformto regulations established by the Food and Drug Administration, whichare available to those in the field. The pharmaceutical composition mayalso be in the form of an aqueous solution containing many of the samesubstances as described above for the reconstitution of a lyophilizedproduct.

The peptide compounds of the presently disclosed inventive concepts canalso be administered as a pharmaceutically acceptable acid- orbase-addition salt, formed by reaction with inorganic acids such ashydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acidssuch as formic acid, acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleicacid, and fumaric acid, or by reaction with an inorganic base such assodium hydroxide, ammonium hydroxide, potassium hydroxide, and organicbases such as mono-, di-, trialkyl and aryl amines, and substitutedethanolamines.

As mentioned above, the peptide compounds of the presently disclosedinventive concepts may be incorporated into pharmaceutical preparationswhich may be used for therapeutic purposes. However, the term“pharmaceutical preparation” is intended in a broader sense herein toinclude preparations containing a peptide composition in accordance withpresently disclosed inventive concepts, used not only for therapeuticpurposes but also for reagent or diagnostic purposes as known in theart. The pharmaceutical preparation intended for therapeutic use shouldcontain a “pharmaceutically acceptable” or “therapeutically effectiveamount” of the peptide compound, i.e., that amount necessary forpreventative or curative health measures. If the pharmaceuticalpreparation is to be employed as a reagent or diagnostic, then it shouldcontain reagent or diagnostic amounts of the peptide compound.

Certain embodiments of the presently disclosed inventive concepts aredirected to any of the compositions described herein above or otherwisecontemplated herein, for use in any of the methods described hereinabove or otherwise contemplated herein.

All of the diagnostic and assay methods listed herein are well withinthe ability of one of ordinary skill in the art given the teachingsprovided herein.

EXAMPLES

The presently disclosed inventive concepts, having now been generallydescribed, will be more readily understood by reference to the followingexamples and embodiments, which are included merely for purposes ofillustration of certain aspects and embodiments of the presentlydisclosed inventive concepts, and are not intended to be limiting. Thefollowing detailed examples and methods describe how to make and use thevarious peptide compounds of the presently disclosed inventive conceptsand are to be construed, as noted above, only as illustrative, and notlimitations of the disclosure in any way whatsoever. Those skilled inthe art will promptly recognize appropriate variations from thecompounds and procedures.

Example 1

Antibacterial Activity of Peptide Compounds Based on Peptide 20-44 (SEQID NO:7) and Method of Enhancing Activity as well as Scale-Up Productionof Compounds Based on Peptides 20-44, 95-122, and 120-146 (SEQ ID NOs:7,26, and 28, respectively).

In certain embodiments, the presently disclosed inventive conceptsinclude peptide compounds, and to compositions thereof, comprisingoligopeptide derivatives comprising an amino acid sequence, and asolubilizing moiety. The oligopeptide derivative may be based on a CAP37protein subsequence selected from, for example, amino acids 20-44 (SEQID NO:7), 23-42 (SEQ ID NO:14), 95-122 (SEQ ID NO:26), 102-122 (SEQ IDNO:27), and 120-146 (SEQ ID NO:28) of CAP37 protein.

In certain embodiments, the presently disclosed inventive conceptsinclude compositions that contain oligopeptide derivatives (i.e.,peptide compounds) that comprise an oligopeptide and a solubilizingmoiety. The oligopeptide derivative/peptide compound is represented byFormula (I) below:S—X_(R)-Pep-Y_(R)wherein Pep is one of SEQ ID NOs:1-14, 25-31, and 47 (see, for example,Table 1) or another appropriate amino acid sequence described herein;X_(R) and Y_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginineresidues, with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginineresidues; and S is a solubilizing moiety comprising 1-10 subunits havingthe formula: NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO—, wherein q=0, 1,2, 3, or 4 and p=1, 2, 3, 4, 5, 6, 7, or 8, R₁ and R₂ are selected fromthe group consisting of H, CH₃ and CH₂CH₃, and n=1 or 2; and with theproviso that S comprises at least one subunit which is not AEEA orAEEEA.

In one non-limiting embodiment, S is from 1-10 AEEP and/or AEEEPsubunits or any other solubilizing subunit disclosed herein linked inany subcombination or order.

As stated herein above, X_(R) and Y_(R) are each independently 0, 1, 2,3, 4, 5, or 6 arginine residues, with the proviso that (X_(R)+Y_(R)) is4, 5, or 6 arginine residues. By way of example, Table 2 below lists thevarious combinations of X_(R) and Y_(R) groups that may be utilized inaccordance with Formula (I). The amino acid sequence RRRR disclosed inTable 2 has been assigned SEQ ID NO:15 herein, whereas the amino acidsequences RRRRR and RRRRRR have been assigned SEQ ID NOs:16 and 17,respectively. In one non-limiting example, X_(R) includes four arginineresidues, while Y_(R) is one arginine residue.

When the peptide compound comprises two cysteines (e.g., SEQ ID NO:7 or14), the oligopeptide may be cyclized via linkage between the twocysteine residues therein.

TABLE 1 Sequence Identifier Compound NamePep Sequences in Accordance with Formula (I) (S-X_(R)-Pep-Y_(R))SEQ ID NO: 1 NQGRHFSGGALIHARFVMTAASCFQ BCC02 (20-44_(ser26))SEQ ID NO: 2 NQGRHFTGGALIHARFVMTAASCFQ 20-44_(thr26) SEQ ID NO: 3NQGRHFMGGALIHARFVMTAASCFQ 20-44_(met26) SEQ ID NO: 4NQGRHFCGGALIHARFVMTAASSFQ BCC03 (20-44_(ser42)) SEQ ID NO: 5NQGRHFCGGALIHARFVMTAASTFQ 20-44_(thr42) SEQ ID NO: 6NQGRHFCGGALIHARFVMTAASMFQ 20-44_(met42) SEQ ID NO: 7NQGRHFCGGALIHARFVMTAASCFQ BCC01 (20-44) SEQ ID NO: 8RHFSGGALIHARFVMTAASC 23-42_(ser26) SEQ ID NO: 9 RHFTGGALIHARFVMTAASC23-42_(thr26) SEQ ID NO: 10 RHFMGGALIHARFVMTAASC 23-42_(met26)SEQ ID NO: 11 RHFCGGALIHARFVMTAASS 23-42_(ser42) SEQ ID NO: 12RHFCGGALIHARFVMTAAST 23-42_(thr42) SEQ ID NO: 13 RHFCGGALIHARFVMTAASM23-42_(met42) SEQ ID NO: 14 RHFCGGALIHARFVMTAASC 23-42Peptide Backbones (X_(R)-Pep-Y_(R)) in Accordance with Formula (I):SEQ ID NO: 18 RRRRNQGRHFSGGALIHARFVMTAASCFQR BCC02-5R (20-44_(ser26))SEQ ID NO: 19 RRRRNQGRHFCGGALIHARFVMTAASSFQR BCC03-5R (20-44_(ser42))SEQ ID NO: 20 RRRRNQGRHFCGGALIHARFVMTAASCFQR BCC01-5R (20-44_(cys))Peptide Compound (S-X_(R)-Pep-Y_(R)) Sequences in Accordance with Formula (I):SEQ ID NO: 21 (AEEP)-(AEEP)-RRRRNQGRHFSGGALIHARFVMTAASCFQR BCC02-5RMPSEQ ID NO: 22 (AEEP)-(AEEP)-RRRRNQGRHFCGGALIHARFVMTAASSFQR BCC03-5RMPSEQ ID NO: 23 (AEEP)-(AEEP)-RRRRNQGRHFCGGALIHARFVMTAASCFQR BCC01-5RMPSEQ ID NO: 24 (AEEP)-(AEEP)-NQGRHFCGGALIHARFVMTAASSFQ BCC03-MPPeptides Based on Listed AA's of CAP37 SEQ ID NO: 26LDREANLTSSVTILPLPLQNATVEAGTR 95-122 SEQ ID NO: 27 TSSVTILPLPLQNATVEAGTR102-122 SEQ ID NO: 28 GTRCQVAGWGSQRSGGRLSRFPRFVNV 120-146QRSEQ ID NO: 29 GTRCQVAGWGSQHSGGRLSRFPRFVNV 120-146QH SEQ ID NO: 30GTRCQVAGWGSWRSGGRLSRFPRFVNV 120-146WR SEQ ID NO: 31GTRCQVAGWGSWHSGGRLSRFPRFVNV 120-146WHPeptide Compound (S-X_(R)-Pep-Y_(R)) Sequences in Accordance with Formula (I):SEQ ID NO: 32 (AEEP)-(AEEP)-RRRRGTRCQVAGWGSQRSGGRLSRFPRFVNVR120-146QR-5RMP SEQ ID NO: 33(AEEP)-(AEEP)-RRRRGTRCQVAGWGSQHSGGRLSRFPRFVNVR 120-146QH-5RMPSEQ ID NO: 34 (AEEP)-(AEEP)-RRRRGTRCQVAGWGSWRSGGRLSRFPRFVNVR120-146WR-5RMP SEQ ID NO: 35(AEEP)-(AEEP)-RRRRGTRCQVAGWGSWHSGGRLSRFPRFVNVR 120-146WH-5RMPSEQ ID NO: 36 (AEEP)-(AEEP)-RRRRNQGRHFTGGALIHARFVMTAASCFQR20-44_(thr26)-5RMP SEQ ID NO: 37(AEEP)-(AEEP)-RRRRNQGRHFMGGALIHARFVMTAASCFQR 20-44_(met26)-5RMPSEQ ID NO: 38 (AEEP)-(AEEP)-RRRRNQGRHFCGGALIHARFVMTAASTFQR20-44_(thr42)-5RMP SEQ ID NO: 39(AEEP)-(AEEP)-RRRRNQGRHFCGGALIHARFVMTAASMFQR 20-44_(met42)-5RMPSEQ ID NO: 40 (AEEP)-(AEEP)-RRRRRHFSGGALIHARFVMTAASCR 23-42_(ser26)-5RMPSEQ ID NO: 41 (AEEP) (AEEP)-RRRRRHFTGGALIHARFVMTAASCR 23-42_(thr26)-5RMPSEQ ID NO: 42 (AEEP)-(AEEP)-RRRRRHFMGGALIHARFVMTAASCR 23-42_(met26)-5RMPSEQ ID NO: 43 (AEEP)-(AEEP)-RRRRRHFCGGALIHARFVMTAASSR 23-42_(ser42)-5RMPSEQ ID NO: 44 (AEEP) (AEEP)-RRRRRHFCGGALIHARFVMTAASTR 23-42_(thr42)-5RMPSEQ ID NO: 45 (AEEP)-(AEEP)-RRRRRHFCGGALIHARFVMTAASMR 23-42_(met42)-5RMPSEQ ID NO: 46 (AEEP)-(AEEP)-RRRRLDREANLTSSVTILPLPLQNATVEAGTRR95-122-5RMP

TABLE 2 Examples of Possible X_(R), Y_(R) Combinations in Formula (I)(S-X_(R)-Pep-Y_(R)) X_(R), Y_(R) Combination Number X_(R) Y_(R) 1 RRRR 2R RRR 3 RR RR 4 RRR R 5 RRRR 6 RRRRR 7 R RRRR 8 RR RRR 9 RRR RR 10 RRRRR 11 RRRRR 12 RRRRRR 13 R RRRRR 14 RR RRRR 15 RRR RRR 16 RRRR RR 17RRRRR R 18 RRRRRR The amino acid sequences RRRR, RRRRR, and RRRRRR havebeen assigned SEQ ID NOs: 15, 16, and 17, respectively.

The peptide compounds of the presently disclosed inventive concepts canbe used, in at least one embodiment, for the treatment of severe Gramnegative infections which include, but are not limited to, Pseudomonasaeruginosa, Salmonella typhimurium, Acinetobacter sp., and E. coli.These organisms are capable of causing fatal hospital-acquiredinfections and are rapidly gaining resistance to current antibiotictherapies. Although the peptide comprising amino acids 20-44 of CAP37(SEQ ID NO:7) has strong antibacterial activity, it is difficult toproduce on a commercial scale, in part because of poor solubility. Usingthe previously established methodology to scale-up this peptide insufficient quantity and purity was unsuccessful. Substantial researchand experimentation, including numerous technical approaches,combinations of amino acids, synthesis procedures, and purifications,were necessary to eventually arrive at the novel active peptidecompounds described herein that can be produced in a commercial scale-upprocedure and thus can be used clinically. Among the novel features ofthe peptide compounds described herein are increased solubility,increased bactericidal efficacy, reduced aggregation, improvedsynthesis, scalability, and increased purity.

The novel peptide compounds, which are derivatized versions of thenative 20-44 peptide, retain substantially all other activities of thenative 20-44 peptide sequence, for example, strong antibacterialactivity, the ability to bind and neutralize lipopolysaccharide (LPS),and negligible cytotoxicity against mammalian cells.

The production of cationic antibacterial peptides which are activeagainst bacteria, exhibit low toxicity to mammalian cells, and that canbe scaled up at a cost of goods that make them commercially viable haseluded most others working in the field. Further, purification can bechallenging due to aggregation. However, the novel peptide compoundsdescribed herein have increased solubility, which will greatlyfacilitate their purification and antibacterial activity.

Other advantages of certain of the novel peptide compounds describedherein include, but are not limited to: (1) their ability to killclinical isolates that have numerous antibiotic resistant patterns; and(2) their bacterial kill rate (for example within minutes) is muchquicker than traditional antibiotics.

The path to scale-up was challenging and not straightforward. Althoughsynthesis of the CAP37 antibiotic peptides in an academic small-scalesetting was successful, this approach was not easily transferable toscale-up production. The major issue appeared to be aggregation.

In the initial stages of the work leading to the presently disclosedinventive concepts, sequential addition of amino acids was used to makethe peptides. Three candidate peptides (CAP37₂₀₋₄₄ (SEQ ID NO:7),CAP37_(20-44ser26) (SEQ ID NO:1), and CAP37_(20-44ser42) (SEQ ID NO:4))were synthesized. All three peptides made by this mode of synthesis wererelatively insoluble. Based on these results, work was focused onproduction of one peptide at a time, and CAP37_(20-44ser42) wasselected. In one embodiment, a fragment Fmoc condensation strategy wasused rather than sequential addition of amino acids. In the fragmentcondensation method, three small fragments were made and condensedtogether to form a CAP37_(20-44ser42) amino acid sequence (SEQ ID NO:4).The rationale behind this reasoning was that three small fragments couldbe synthesized in a highly purified form and linked together, therebyovercoming some of the aggregation problems. The product from thissynthesis was dialyzed against 1% acetic acid and lyophilized anddetermined to be approximately 50% pure. Antimicrobial activity of thispeptide was approximately half of what was normally obtained. In oneembodiment, the dialysis procedure was changed to employ a dilutetri-fluoro acetic acid (TFA) solution (e.g., 0.1%), which obtained aproduct with stronger antimicrobial activity and greater purity (about80%). In the next production, a cysteine residue was used that wasprotected during synthesis and then de-protected in the final stages ofpurification, in an attempt to improve stability.

Eventually, 20 grams of each compound was produced; however, the majortechnical issues of aggregation and solubility, although overcome to alarge degree, still remained, thus indicating that large scaleproduction for clinical trials may not be feasible. After many trialsand experiments, the idea of adding a plurality of arginine (R) residuesat the COOH-terminus and/or the NH₂-terminus of the peptide wasdiscovered. In the first iteration, three R residues were added, one atthe COOH-terminus and two at the NH₂ terminus. The effect on solubilityand activity by using arginine residues was markedly improved. Based onthis successful result, each of the three sequences was synthesized as apeptide backbone having a total of five arginine residues (e.g., one atthe COOH-terminus and four at the NH₂-terminus, e.g., see SEQ IDNOs:18-20 in Table 1). Scale-up of the compounds was successful,enabling synthesis of gram quantities of each peptide. Moreover, the invitro antibacterial activity was significantly greater than what hadbeen previously observed with the original peptides. The three5-arginine peptides showed >97% kill at concentrations as low as 2.5 μM,which is 10-fold higher than what had been observed previously. Allthree 5-arginine peptides (SEQ ID NOs:18-20) also showed potent activityagainst other Gram negative organisms including Escherichia coli andSalmonella typhimurium. Due to the increased potency of these newlysynthesized peptides, it was queried whether the peptides may havecytotoxicity on mammalian cells. The peptides were evaluated using theLactic Dehydrogenase Cytotoxicity Detection kit (Roche DiagnosticsCorp., Indianapolis, Ind.), and it was found that the peptides hadminimal cytotoxic activity even at the highest concentrations tested (75μM). After 4 hours incubation, all three peptides showed <4%cytotoxicity. After 24 hours incubation, there was a slight increase incytotoxicity levels; peptide 20-44_(ser26) (SEQ ID NO:18) showed 23%,20-44_(ser42) (SEQ ID NO:19) showed 5%, and 20-44_(cys) (SEQ ID NO:20)showed 10% activity.

All three 5-arginine peptides (SEQ ID NOs:18-20) bound Pseudomonas LPS,as measured by the Limulus Amebocyte Lysate (LAL) assay. However, it hadnot been demonstrated that these peptides could neutralize the toxiceffects of LPS. The well-characterized response of RAW264.7 macrophagecells was used to produce tumor necrosis-factor-alpha (TNF-α) inresponse to LPS to determine whether the peptides could dampen therelease of this cytokine. All three 5-arginine peptides attenuated therelease of TNF-α in response to Pseudomonas LPS; and this result wasdose dependent. Importantly, pre-treatment (3 hours prior to LPSaddition) as well as post-treatment of the RAW264.7 cells (3 hours postLPS addition) with peptide dampened the cytokine release. A controlpeptide was unable to attenuate the release of TNF-α. Further studiesshowed that this attenuation was most likely due to blocking of theactivity of the transcription factor nuclear factor-kappa B (NF-κB). Thefinding that the novel peptides bind and neutralize the effect ofPseudomonas LPS is of critical importance since this ensures that thenovel therapeutic peptide compounds described herein will not only killthe bacteria, but will also neutralize the LPS endotoxin released by thebacteria which plays such an important role in the rapid progression ofsepsis.

When the arginines were linked to the peptide to form the peptidebackbone (SEQ ID NOs:18-20), there was marked increase in solubility ofthe peptides and very potent activity (capable of reducing bacterialload from 1×10⁶ CFU/ml to 1×10 CFU/ml, a 5 log reduction). However,although these peptides (SEQ ID NOs:18-20) were highly potent, theycould not be purified beyond about 70% purity. Much time andexperimental effort was spent in the laboratory exploring purificationtechniques, but yield and purity could not be increased beyond about70%. This was extremely disappointing, since a large number of in vitroLPS binding experiments and bactericidal studies had been performed todemonstrate efficacy. Finally, after extensive additionalexperimentation, another solubilizing moiety was added to the peptides,and this addition increased half-life, solubility, and purity of thepeptides. In this embodiment, the solubilizing moiety comprised a pairof small (“mini”) PEG molecules AEEP, each having a molecular weight ofabout 160.

The two PEG solubilizing moieties comprising AEEP were linked end-to-endto the amino terminal end of the peptide backbone. Table 1 shows thethree peptide compounds comprising two AEEP molecules linked to each ofSEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 and designated therein bythe compound names BCC02-5RMP (SEQ ID NO:21), BCC03-5RMP (SEQ ID NO:22),and BCC01-5RMP (SEQ ID NO:23), respectively. In these 3 peptidecompounds (referring to Formula (I) described above), S is AEEP-AEEP,X_(R) is RRRR (SEQ ID NO:15), Y_(R) is R, and Pep is SEQ ID NO:1, SEQ IDNO:4, and SEQ ID NO:7, respectively.

Purity of these peptides with the 5 arginines and the two AEEA moietiesrange consistently from 96.12% to 98.62%. Good laboratory practice (GLP)grade peptide synthesis has been achieved with BCC02-5RMP (SEQ ID NO:21), and 75 grams of peptide have been synthesized at 97.24% purity.

In addition to peptides based on the 20-44 sequence (SEQ ID NO: 21 toSEQ ID NO: 23), the inclusion of the five arginine residues and the twosmall PEG (AEEP) molecules during the synthesis of the peptides based onamino acids 95-122 (SEQ ID NO: 26 and SEQ ID NO: 27) and 120-146 (SEQ IDNOs: 32-35) will aid in the overall scale-up of these peptides as well.

In vitro bactericidal activity: The three peptide compounds BCC01-5RMP(SEQ ID NO:23), BCC02-5RMP (SEQ ID NO:21), and BCC03-5RMP (SEQ ID NO:22)were assayed for in vitro bactericidal activity against Pseudomonasaeruginosa, Escherichia coli, Salmonella typhimurium, and Acinetobacterbaumannii. The starting inoculum for all experiments was 1×10⁶ colonyforming units (CFU)/ml. The CFU remaining at the end of 60 to 180 minuteincubation was determined, and the data plotted as a log reduction inCFU/ml. The peptides were most active against Pseudomonas andAcinetobacter with strong, but lesser activity against Salmonella and E.coli. Table 3 shows activity of the compounds against Pseudomonas. Thein vitro bactericidal activity shown in Table 3 indicates thatBCC03-5RMP (SEQ ID NO:22) has a slight edge over the other two peptidesin this assessment.

TABLE 3 P. aeruginosa ATCC 27853 % kill BCC02-5RMP (SEQ ID NO: 21)Peptide Experi- Experi- Experi- Experi- concentration ment #1 ment #2ment #3 ment #4   50 μg/ml 100 100 100 100   25 μg/ml 100 99.98 100 10012.5 μg/ml 98.21 90.78 99.93 99.96 6.25 μg/ml Not done Not done 98.8599.26 P. aeruginosa ATCC 27853 % Kill BCC03-5RMP (SEQ ID NO: 22) PeptideExperi- Experi- Experi- Experi- concentration ment #1 ment #2 ment #3ment #4   50 μg/ml 100 Not done Not done Not done   25 μg/ml 100 100 100100 12.5 μg/ml 99.91 99.98 100 99.56 6.25 μg/ml Not done 99.34 99.9398.32 P. aeruginosa ATCC 27853 % Kill BCC01-5RMP (SEQ ID NO: 23) PeptideExperi- concentration ment #1 50 μg/ml 100 25 μg/ml 99.83 12.5 μg/ml  79.21

Tables 4-6 show the results of various peptides and peptide compoundsagainst P. aeruginosa. Table 4 shows the results of an investigation ofthe effects of peptide or peptide compounds BCC01-5R (SEQ ID NO: 20),BCC01-5RMP (SEQ ID NO: 23), BCC02-5R (SEQ ID NO: 18), BCC02-5RMP (SEQ IDNO: 21), BCC03-5R (SEQ ID NO: 19), BCC03-5RMP (SEQ ID NO:22), andBCC03-MP (SEQ ID NO:24) against P. aeruginosa ATCC® strain 27853′(American Type Culture Collection, Manassas, Va.). A comparison ofactivity BCC03-5R (SEQ ID NO: 19) and BCC03-5RMP (SEQ ID NO: 22), thetwo peptide compounds with the additional five arginine residues versusBCC03-MP (SEQ ID NO: 24) (i.e., the peptide SEQ ID NO:4 which has thesolubilizing moiety S attached thereto but does not have the fiveadditional R residues) shows that the inclusion of the 5 argininesenhance bactericidal activity. Table 4 further compares the activity ofseveral other compounds, including the “5R” versions of BCC01, i.e., SEQID NO:20 (which comprises SEQ ID NO: 7 with five additional arginines),the “5R” version of BCC02 (i.e., SEQ ID NO:18, which comprises SEQ IDNO: 1 with five additional arginines), and the 5RMP versions of BCC01(SEQ ID NO: 23) and BCC02 (SEQ ID NO: 21). The data generally indicatethe superiority of BCC03-5RMP (SEQ ID NO:22) and BCC02-5RMP (SEQ ID NO:21) and the superiority of the 5R or 5RMP versions of the peptide overthe “AEEP only” peptide against P. aeruginosa. Table 5 shows the strongactivity of BCC03-5RMP (SEQ ID NO: 22) against a number of clinicalisolates of Pseudomonas obtained from infected skin wounds with variousantibiotic resistant profiles. Table 6 shows the strong in vitrobactericidal activity of BCC02-5RMP (SEQ ID NO: 21) against clinicalisolates of Pseudomonas aeruginosa obtained from patients with bacterialkeratitis.

In vivo bactericidal activity of BCC02-5RMP (SEQ ID NO:21): FIG. 1 showsresults of the use of BCC02-5RMP (SEQ ID NO:21) in vivo in a bacterialkeratitis model in C57/BL6 mice. A circular wound (2 mm) was created onthe mouse cornea by removing the epithelium and infecting the wound with10⁵ CFU of P. aeruginosa (ATCC® 27853′ (American Type CultureCollection, Manassas, Va.)). Infected wounds were treated with saline orsaline containing indicated concentrations of peptide every 15 minutesfor 2 hours, then every 30 minutes for 3 hours on the first day.Infected wounds were treated twice on the second day and once on thethird day. Mice were killed at 48 hours post-infection, and the colonyforming units per eye (CFU/eye) were quantified. The means are plottedand are representative of 5 to 9 mice per group. Mann Whitney test wasperformed for each group as compared to the saline control group. Thepeptide compound effectively killed all bacteria and cured the infectionat concentrations ranging between 0.25 and 0.75 mg/ml.

TABLE 4 5*10³ CFU/ml unless Italicized = Percent Kill and Log Reduction1*10⁶ CFU/ml BCC01 BCC02 BCC03 BCC01 5R BCC01 5R MP BCC02 5R BCC02 5R MPBCC03 5R BCC03 5R MP BCC03 MP Lot #026C09 Lot #J918 Lot #028C09 Lot#J880 Lot #027C09 Lot #J971 Lot #130L09 SEQ ID NO: 20 SEQ ID NO: 23 SEQID NO: 18 SEQ ID NO: 21 SEQ ID NO:19 SEQ ID NO: 22 SEQ ID NO: 24Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas PseudomonasPseudomonas aeruginosa aeruginosa aeruginosa aeruginosa aeruginosaaeruginosa aeruginosa Peptide ATCC 27853 ATCC 27853 ATCC 27853 ATCC27853 ATCC 27853 ATCC 27853 ATCC 27853 Concen- Log Log Log Log Log LogLog tration Percent Reduc- Percent Reduc- Percent Reduc- Percent Reduc-Percent Reduc- Percent Reduc- Percent Reduc- μg/mL Kill tion Kill tionKill tion Kill tion Kill tion Kill tion Kill tion 400 92.8 0.4 200 100.03.1 74.4 −0.1 100 100.0 3.1 44.0 −0.3 87.6 100.0 2.4 100.0 2.4 100.0 2.450 100.0 5.5 100.0 6.5 100.0 3.1 −2.9 −0.4 25 99.8 3.0 100.0 6.0 100.03.1 100.0 7.0 −27.3 −0.5 12.5 79.2 0.5 100.0 3.0 100.0 3.1 100.0 5.0−45.6 −0.5 8.76 99.5 1.8 99.6 1.8 99.1 1.4 6.25 99.3 1.5 100.0 3.1 99.932.5 −38.2 −0.5 3.125 2.19 87.9 0.4 87.0 0.8 77.1 0.2 1.56 0.78 0.547563.2 0.0 75.6 0.1 65.1 0.0 0.137 25.4 −0.3 24.9 −0.3 33.8 −0.2

TABLE 5 Pseudomonas aeruginosa clinical isolates from infected skinwounds Isolate Isolate Isolate Isolate #97-1 #98-1 #99-1 #100-1 PeptideBCC03-5RMP (SEQ ID NO: 22) concen- Expt Expt Expt Expt Expt Expt ExptExpt tration #1 #2 #1 #2 #1 #2 #1 #2 25 99.99 100 99.42 100 100 100 100100 μg/ml 12.5 99.66 100 98.47 99.99 100 100 99.98 99.99 μg/ml

TABLE 6 Pseudomonas aeruginosa clinical isolates from bacterialkeratitis patients Isolate #5 Isolate #6 Isolate #18 Isolate #46 PeptideBCC02 5RMP (SEQ ID NO: 21) concen- Expt # Expt # Expt # Expt # tration 12 3 1 2 3 1 2 3 1 2 3   25 μg/ml 100 99.99 100 100 100 100 100 100 100100 100 100 12.5 μg/ml 99.97 99.77 99.91 100 99.1 99.98 100 99.88 99.99100 99.95 99.99 Pseudomonas aeruginosa clinical isolates from bacterialkeratitis patients Isolate #47 Isolate #67 BCC02 5RMP (SEQ ID NO: 21)Peptide Expt # concentration 1 2 3 1 2 3   25 μg/ml 100 100 100 10099.99 100 12.5 μg/ml 99.9 99.76 99.97 99.99 99.55 99.64

Example 2 In Vitro LPS Binding and Neutralization

BCC01-5RMP (SEQ ID NO:23), BCC02-5RMP (SEQ ID NO:21), and BCC03-5RMP(SEQ ID NO:22) were tested for their ability to bind lipopolysaccharide(LPS) using the limulus amebocyte lysate method (LAL). All threepeptides bound Pseudomonas LPS. There was no statistical difference intheir ability to bind LPS using the LAL technique. The ability of thepeptides to neutralize LPS was assessed by determining their ability toattenuate the release of tumor necrosis factor-alpha (TNF-α) fromLPS-stimulated RAW264.7 cells (a mouse macrophage cell line). All threepeptides were seen to attenuate the release of TNF-α. No significantdifferences were observed between the three peptides to neutralize LPSas measured by cytokine release. Peptides based on sequence 120-146 (SEQID NOs:28-31) also bind and neutralize Pseudomonas LPS.

Example 3 Synthesis and Purification of BCC03-5RMP

The purpose of this synthesis was to synthesize the peptide BCC03-5R(SEQ ID NO:19) and couple it to two small PEG molecules (AEEP) toproduce BCC03-5RMP (SEQ ID NO:22):(AEEP)-(AEEP)-RRRRNQGRHFCGGALIHARFVMTAASSFQR. After the synthesis, thepeptide compound was purified and lyophilized. As noted previously, inthe compound name BCC03-5RMP, “R” refers to arginine and “MP” refers toa solubilizing group S made up of two AEEP moieties.

Synthesis: The peptide compound may be synthesized using solid phasepeptide synthesis using Fmoc chemistry protocol. The peptide chain wassynthesized on Fmoc-Arg(Pbf)-Wang resin. Three equivalents of aminoacids were used for each coupling, and couplings were performed usingthe DIC/HOBT method. After complete synthesis of the peptide chain, bothFmoc-mini-PEG™s were coupled by the DIC/HOBT method. After thesynthesis, the resin was washed and dried.

The peptide compound was deprotected and cleaved from the resin using acocktail of TFA-containing scavengers. Resin was filtered off, andfiltrate was evaporated on an evaporator to remove TFA. The peptidecompound was precipitated with ether; precipitate was filtrated off anddried under reduced pressure to get crude peptide.

Purification: The purification may be performed by RP-HPLC using the YMCODS Gel C18 as support. 0.1% TFA/H₂O and acetonitrile were used assolvents for purification. The fractions were checked by analyticalHPLC, and fractions with required purity were pooled together. The poolwas evaporated and lyophilized. Purity of peptides generated using thistechnique was consistently >95%.

In other embodiments of the presently disclosed inventive concepts, thepeptide compounds comprise derivatives of other amino acid portions ofthe CAP37 protein, including, but not limited to, peptides 23-42,95-122, 102-122, and 120-146 which have been derivatized withsubstitutions (e.g., at positions 131 and/or 132), and/or with N- and/orC-terminal arginine residues and small PEG molecules as describedelsewhere herein, such as in Formula (I) and Table 1 and accompanyingdescription elsewhere herein. Such peptides 23-42, 95-122, 102-122, and120-146 of CAP37 protein are described for example in U.S. Pat. Nos.5,107,460; 7,354,900; and 7,893,027; which are hereby expresslyincorporated by reference herein in their entireties.

The “Pep” sequence of the Formula (I) peptide compound described hereinmay comprise the following sequence (SEQ ID NO:25):R—H—X₃—X₄—X₅—X₆—X₇—X₈—X₉—H—X₁₁—R—X₁₃—X₁₄-M-X₁₆—X₁₇—X₁₈—X₁₉—X₂₀wherein X₃ and X₁₃ are phenylalanine, tyrosine, arginine, lysine orhistidine; X₄ is selected from cysteine, serine, threonine, andmethionine; X₅ and X₆ are selected from glycine and alanine; X₇, X₁₁,and X₁₄ are selected from alanine, leucine, isoleucine, and valine; X₉,X₁₇, and X₁₈ are selected from alanine, leucine, isoleucine, and valine;X₁₆ is selected from serine, threonine, and methionine; X₁₉ is selectedfrom serine, threonine and methionine; X₂₀ is selected from cysteine,serine, and methionine; R is arginine; H is histidine; and M ismethionine. This sequence is a derivative of SEQ ID NO:14 (i.e., aminoacids 23-42 of CAP37 protein).

Similarly, the “Pep” sequence of the Formula (I) peptide compounddescribed herein may comprise the following sequence (SEQ ID NO:47):R—H—X₃—X₄—X₅—X₆—X₇—X₈—X₉—H—X₁₁—R—X₁₃—X₁₄-M-X₁₆—X₁₇—X₁₈—X₁₉—X₂₀wherein X₃ and X₁₃ are phenylalanine, tyrosine, arginine, lysine, orhistidine; X₄ is selected from cysteine, serine, threonine, andmethionine; X₅ and X₆ are selected from glycine and alanine; X₇, X₁₁,and X₁₄ are selected from alanine, leucine, isoleucine, and valine; X₉,X₁₇, and X₁₈ are selected from alanine, leucine, isoleucine, and valine;X₁₆ is selected from serine, threonine, and methionine; X₁₉ is selectedfrom serine, threonine, and methionine; X₂₀ is selected from cysteine,serine, threonine, and methionine; R is arginine; H is histidine; and Mis methionine.

Example 4

Chemotactic and Wound Healing Activity of 95-122-Based (SEQ ID NOs:26-27and 46) and 120-146-Based (SEQ ID NOs:28-35) Peptide Compounds

In one embodiment, certain peptide compounds disclosed herein includingthose comprising SEQ ID NOs:26-35 have chemotactic activity for hostcells including monocytes and corneal epithelial cells. For example,peptide 95-122 is shown herein to promote corneal wound healing in an invivo mouse model of corneal epithelial abrasion and dermal skin woundhealing in swine. Peptide compounds based on peptide 120-146 of CAP37protein also have bacterial activity as well as immune regulation ofhost cells. Non-limiting examples of such peptide compounds aredescribed below and are listed in Table 1 (see SEQ ID NOs:26-35).

Peptide compounds of the presently disclosed inventive concepts, such asbut not limited to those mentioned below, can be used as therapeutics inaiding the rapid healing of wounds. Although antibiotics exist that canbe used to treat infected wounds, none of these agents has been shown toboth kill bacteria and accelerate wound healing and/or improve skingraft acceptance. Peptide compounds such as those described herein thatcan do both will have a great benefit medically and thus have strongcommercial potential. Among the types of wounds that can be treatedusing these peptide compounds include, but are not limited to, chronic“non-healing” wounds such as diabetic ulcers, “bed sores,” burnspatients, infected psoriasis lesions, “dry eye” inflammatory conditions,as well as acute wounds such as ocular ulcers, ocular wounds, and woundsof skin and epithelial tissues. As noted, another function of thepeptide compounds is promotion of the healing and acceptance of graftssuch as skin grafts.

Peptides 95-122 and 102-122: Peptide 95-122 (LDREANLTSSVTILPLPLQNATVEAGTR; SEQ ID NO:26) corresponds to amino acids 95-122 of CAP37 protein.Peptide 102-122 (TSSVTILPLPLQNATVEAGTR; SEQ ID NO:27) is a truncation of95-122 and corresponds to amino acids 102-122 of CAP37 protein. Thesepeptides and derivatives thereof can be used intherapeutically-effective amounts to, for example, increaseproliferation and migration of corneal epithelial cells in a subjecthaving a corneal ulcer or wound and increasing their adherence and inaccelerating dermal wound healing.

These peptides (as shown, for example, in FIGS. 2-6 for Peptide 95-122),can be used, for example, to cause and/or promote: (a) chemotaxis ofmonocytes; (b) chemotaxis of human epithelial cells such as cornealepithelial cells; (c) wound healing in corneal epithelia (FIGS. 2 and3). FIG. 2 is data analyzed and represented as histogram. FIG. 3 is theactual photograph of a treated mouse eye. Data from this photograph wasused to generate the histogram in FIG. 2; and (d) cytokine production inresponse to intrastromal injection of the peptides (FIGS. 4-6).

“Peptide 120-146” (and Derivatives): Described below are severalpeptides based on Peptide 120-146 (SEQ ID NO:28), also known as120-146QR. These peptides include, but are not limited to, 120-146QH,120-146WR, 120-146WH, 120-146QR-5RMP, 120-146 QH-5RMP, 120-146WR-5RMP,and 120-146WH-5RMP.

1. Peptide 120-146, also referred to herein as peptide 120-146QR for theglutamine (Q) at position 131 and arginine (R) at position 132, has thefollowing sequence: GTRCQVAGWGSQRSGGRLSRFPRFVNV (SEQ ID NO:28).

2. Peptide 120-146QH is derived from an induced form of CAP37 proteinthat was sequenced from corneal epithelial cells and has the followingsequence, which is the same as peptide 120-146QR except that thearginine at position 132 is replaced by a histidine (hence the name120-146QH): GTRCQVAGWGSQHSGGRLSRFPRFVNV (SEQ ID NO:29).

3. Peptide 120-146WR is an analog of peptide 120-146QR wherein theGlutamine (Gln, Q) at position 131 has been replaced by a tryptophan(Trp,W). This peptide has the following sequence:GTRCQVAGWGSWRSGGRLSRFPRFVNV (SEQ ID NO:30).

4. Peptide 120-146WH is an analog of peptide 120-146QH which has had theglutamine at position 131 replaced by a tryptophan (Trp, W). Thispeptide has the following sequence: GTRCQVAGWGSWHSGGRLSRFPRFVNV (SEQ IDNO:31).

5. Peptide 120-146QR-5RMP is a derivatized version of peptide 120-146QRwhich includes 2 small PEG (AEEP) moieties (“MP”) and 4 arginine (R)residues at the amino terminus, and one R at the carboxy terminus. Thispeptide has the following sequence:(AEEP)-(AEEP)-RRRRGTRCQVAGWGSQRSGGRLSRFPRFVNVR (SEQ ID NO:32).

6. Peptide 120-146QH-5RMP is a derivatized version of peptide 120-146QHwhich includes 2 small PEG (AEEP) moieties (“MP”) and 4 arginine (R)residues at the amino terminus, and one R at the carboxy terminus. Thispeptide has the following sequence:(AEEP)-(AEEP)-RRRRGTRCQVAGWGSQHSGGRLSRFPRFVNVR (SEQ ID NO:33).

7. Peptide 120-146WR-5RMP is a derivatized version of peptide 120-146WRwhich includes 2 small PEG (AEEP) moieties (“MP”) and 4 arginine (R)residues at the amino terminus, and one R at the carboxy terminus. Thispeptide has the following sequence:(AEEP)-(AEEP)-RRRRGTRCQVAGWGSWRSGGRLSRFPRFVNVR (SEQ ID NO:34).

8. Peptide 120-146WH-5RMP is a derivatized version of peptide 120-146WHwhich includes 2 small PEG (AEEP) moieties (“MP”) and 4 arginine (R)residues at the amino terminus, and one R at the carboxy terminus. Thispeptide has the following sequence:(AEEP)-(AEEP)-RRRRGTRCQVAGWGSWHSGGRLSRFPRFVNVR (SEQ ID NO:35).

Materials and Methods of Example 4

Synthesis of peptides: Peptides were synthesized using solid phasesynthesis on an Applied Biosystems model 430A peptide synthesizer(0.1-mmol or 0.5-mmol scale).

Animals: C57BL/6 female mice were purchased from The Jackson Laboratory(Bar Harbor, Me., USA). All animals were treated humanely. TheInstitutional Animal Care and Use Committee (IACUC) at the University ofOklahoma, Oklahoma City, Okla. and the Dean McGee Eye Institute,Oklahoma City, Okla., approved all animal research protocols.

In vivo model of corneal wound healing: The in vivo model of woundhealing was carried out using a disposable biopsy punch (2 mm, Miltex,York, Pa.) to demarcate the mouse cornea and a 0.5 mm burr using theAlgerBrush II (The Alger Company, Inc., Lago Vista, Tex.) to remove thecorneal epithelium. The corneal abrasions were treated at 0 and 16 hourswith peptide 95-122 (10⁻⁵ M), peptide 120-146WH (10⁻⁶ M and 10⁻⁸ M),peptide 120-146WR ((10⁻⁶ M and 10⁻⁸ M), or vehicle control (0.9% sodiumchloride, pH 5.5, Baxter, Deerfield, Ill.). Corneal abrasions werevisualized using sterile fluorescein sodium ophthalmic strips USP(Fluorets®, Chauvin Laboratory, Aubenas, France) dampened with sterilePBS. Images were taken at 0, 16, and 24 hours immediately followingfluorescein staining.

Chemotaxis: Chemotaxis assays were performed using the modified Boydenchemotaxis chamber assay. Peptide 120-146QH was used at 10⁻⁴ M, 10⁻⁶ M,10⁻⁸ M, 10⁻⁶ M, and 10⁻¹²M, and peptide 120-146QR was used at 10⁻⁴M,10⁻⁶M, 10⁻⁸M, and 10⁻¹⁰ M.

Statistical analysis: In vivo wound healing experiments were analyzedusing an unpaired t-test and ANOVA. Boyden chamber chemotaxisexperiments were analyzed using a Wilcoxon signed-rank test. Statisticswere calculated using GraphPad Prism 4.03 (GraphPad Software, Inc., SanDiego, Calif.). The means of independent experimental values areshown±SEM and a P value of <0.05 was considered significant for allstatistical analyses.

Results of Example 4

Peptides 120-146QR (SEQ ID NO:28) and 120-146QH (SEQ ID NO:29)facilitate chemotaxis in HCECs. To elucidate the effect of CAP37-derivedpeptides on HCEC migration, HCECs were treated with peptides 120-146QH(SEQ ID NO:29), based on the native CAP37 sequence found in HCECs, and120-146QR (SEQ ID NO:28), based on the native CAP37 sequence found inneutrophils, and migration in response to these peptides was measuredusing the modified Boyden chemotaxis chamber assay. Treatment withpeptide 120-146QH (SEQ ID NO:29) at 10⁻⁴ M, 10⁻⁶ M, 10⁻⁸ M, and 10⁻¹⁰ Mand with peptide 120-146QR (SEQ ID NO:28) at 10⁻⁶ M and 10⁻⁸ M was foundto significantly increase migration of HCECs in a dose-dependent manner(FIG. 7). Both peptides maximally facilitated migration between 10⁻⁶Mand 10⁻⁸ M. There was a significant increase in migration in response toHB-EGF (positive control) and CAP37 (positive control for the peptide)(FIG. 7). The migration with 120-146QH (SEQ ID NO:29) at 10⁻⁶ M and 10⁻⁸M was comparable to the migration obtained with the whole CAP37 protein.Levels of migration with 120-146QR (SEQ ID NO:28) were less than themigration obtained with the whole protein, but significant when comparedto the buffer control.

Peptide 120-146WH (SEQ ID NO:31) facilitates corneal epithelial woundhealing. To determine the effect of both 120-146WR (SEQ ID NO:30) and120-146WH (SEQ ID NO:31) peptides on corneal wound healing, an in vivomodel of wound healing was utilized. Peptide 120-146WH (SEQ ID NO:31)contributed to the healing of in vivo wounds in a dose-dependent manner(FIGS. 8 and 9).

Results showed that peptide 120-146WH (SEQ ID NO:31) maximallyfacilitated wound healing between 10⁻⁶ M and 10⁻⁸ M. The amount ofhealing in 120-146WH-treated wounds was significantly greater(***P<0.001 and *P<0.05 as determined by unpaired t-test) thanvehicle-treated samples (FIG. 8). Representative images of the in vivowounds show a dose-dependent increase in wound healing in the120-146WH-treated wounds versus the vehicle treated wounds (FIG. 9).Data presented herein indicate that the 120-146-based peptide compoundsnot only kill bacteria but also mediate chemotaxis and promote woundhealing. None of the other bioactive peptides based on residues 20-44(SEQ ID NO:7) and 95-122 (SEQ ID NO:26) of the native CAP37 protein havethis dual function.

Derivatives of peptide 120-146 comprise, in one embodiment, formulationssuitable for dermal and or ophthalmic use, and in certain embodiments,formulations for wound healing are effective in wound healing (FIGS. 7-9show, for example, 120-146-based peptides (e.g., SEQ ID NOs: 28-31).Certain embodiments of the presently disclosed inventive conceptsfurther include combinations of a therapeutic using a presentlydisclosed peptide compound with wound healing properties along withanother of the 120-146-based peptide compounds or any of the 20-44-5RMPseries of CAP37 peptide compounds (i.e., SEQ ID NOs:21-23) to provideboth healing as well as anti-infective power.

Example 5 Antibiotic Activity of 120-146-Based Peptide Compounds

In this Example, peptide compounds based on Peptide 120-146 (i.e., SEQID NOS:28-35), as well as compositions containing these compounds, wereshown to possess antibacterial activity against various bacteria. ThisExample demonstrates the usefulness of these compounds/compositions asantibiotics for treating various infections, including but not limitedto, Pseudomonas aeruginosa, Acinetobacter baumannii, and other bacteria(see FIGS. 10-14). These peptide compounds/compositions can be used inmonotherapy against bacteria that are resistant to standard antibiotics;these peptide compounds/compositions can also be used as antibioticswhich are effective at neutral pH (FIGS. 11-12), and which are alsoactive at low pH, including levels where gentamicin is not effective(FIGS. 10 and 13).

Materials and Methods of Example 5

For FIGS. 10-13, in vitro bactericidal assays were performed using astarting inoculum of 1×10⁶ CFU/ml of the bacterial suspension. The CFUremaining at the end of the 60-180 minute incubation was determined, andthe data was plotted as log reduction in CFU/ml.

In particular, each of the test peptides (CAP37 peptides 120-146WR,120-146WH, and 95-122 (SEQ ID NOs:30, 31, and 26, respectively)) wasused at 25, 12.5, 6.25, and 3.12 μM. Gentamicin was used at 4 μg/ml.1×10⁶/ml Acinetobacter baumannii ATCC® BAA-747™ or Pseudomonasaeruginosa ATCC® 27853™ (American Type Culture Collection, Manassas,Va.) were incubated with the peptides, negative buffer control (tryptonesaline), and positive control gentamicin at pH 5.5 or 7.2. A 60 minuteincubation was used for Acinetobacter baumannii ATCC® BAA-747™, while a180 minute incubation was used for Pseudomonas aeruginosa ATCC®27853™/50 μl aliquots were plated out on agar plates and incubatedovernight. Colony forming units were counted after overnight incubationand CFU/ml calculated.

For FIG. 14, 1×10⁶/ml Pseudomonas aeruginosa ATCC® 27853™ were incubatedwith each of the test peptides (CAP37 peptides 120-146WR, 120-146WH,120-146QR, 120-146QH, 120-146QR-5RMP, and 120-146QH-5RMP (SEQ ID NOs:30,31, 28, 29, 32, and 33, respectively)) at 150, 75, and 37.5 μg/ml, ornegative buffer control (tryptone saline), for 180 minutes at pH 5.5. 50μl aliquots were plated out on agar plates and incubated overnight.Colony forming units were counted after overnight incubation and CFU/mlcalculated.

For FIGS. 15-17, in vivo bactericidal activity of the peptides was alsoinvestigated using the standard Pseudomonas keratitis model in the mouseeye (FIGS. 15-17). Peptides 120-146QR-5RMP (SEQ ID NO:32), 120-146WH(SEQ ID NO:31), and 120-146WR (SEQ ID NO: 30) were applied topically atdoses of 2, 5, 10, and 20 mg/ml to the wounded cornea that had beeninfected with 10⁵ CFU of Pseudomonas aeruginosa. The efficacies of thepeptides were compared to treatment with the vehicle control. Mice wereeuthanized at 48 hours post infection, and the CFU/eye were quantified.

Results of Example 5

FIGS. 10 and 11 show bactericidal activity of CAP37 peptides 120-146WR(SEQ ID NO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26)against Acinetobacter baumannii ATCC® BAA-747™ (American Type CultureCollection, Manassas, Va.) at two different pH's, and as compared withgentamicin as the comparator antibiotic. As can be seen, the CFU/ml werereduced by almost 4 logs at pH 5.5 (FIG. 10) and by almost 5.5 logs atpH 7.2 (FIG. 11) at the highest peptide concentrations of the CAP37peptides 120-146WR and 120-146WH used; thus, the peptides had greateractivity at pH 7.2 than at pH 5.5. The standard antibiotic gentamicinwas unable to kill the bacteria at pH 5.5, even though it was used atmore than its standard minimum inhibitory concentrations (MIC). Incontrast, the standard antibiotic gentamicin was active against thebacteria at pH 7.2. Peptide 95-122 was not active against Acinetobacterat either pH. Peptides 120-146WR and 120-146WH at 25 μM and peptide120-146WR at 12.5 μM were highly active against A. baumannii.

FIGS. 12 and 13 show bactericidal activity of CAP37 peptides 120-146WR(SEQ ID NO:30), 120-146WH (SEQ ID NO:31), and 95-122 (SEQ ID NO:26)against Pseudomonas aeruginosa ATCC® 27853′ (American Type CultureCollection, Manassas, Va.) at two different pH's, and as compared withgentamicin as the comparator antibiotic. As can be seen, the CFU/ml werereduced by 6 logs at pH 7.2 (FIG. 12) and by 4-5 logs at pH 5.5 (FIG.13) at the highest concentrations of peptides 120-146WR and 120-146WHused; thus, the peptides had greater activity at pH 7.2 than at pH 5.5.Peptides 120-146WR and 120-146WH at 25 μM were as effective as thestandard antibiotic gentamicin at pH 7.2; gentamicin was not aseffective as the peptides against the bacteria at pH 5.5 (showing only a2 log reduction), even though it was used at more than its standard MIC.Peptide 95-122 was not active against Pseudomonas aeruginosa at eitherpH. Peptides 120-146WH at 25 μM and 120-146WR at 12.5 μM had the sameeffect. Peptide 120-146WR at 25 μM and gentamicin were highly activeagainst P. aeruginosa.

FIG. 14 is a comparison of bactericidal activity of CAP37 peptides120-146WR (SEQ ID NO:30), 120-146WH (SEQ ID NO:31), 120-146QR (SEQ IDNO:28), 120-146QH (SEQ ID NO:29), 120-146QR-5RMP (SEQ ID NO:32), and120-146QH-5RMP (SEQ ID NO:33) for Pseudomonas aeruginosa ATCC® 27853™(American Type Culture Collection, Manassas, Va.). As can be seen at thehighest concentrations of peptides with the native sequence (120-146QRand 120-146QH, SEQ ID NOs:28 and 29, respectively), the CFU count waslowered by 1-2 logs. With the replacement of the amino acid residue atposition 131 with a tryptophan, the killing efficiency of the peptides(120-146WR and 120-146WH, SEQ ID NOs:30 and 31, respectively) wasincreased, reducing the CFU by 6 logs. Modification of the nativesequence (120-146QR-5RMP and 120-146QH-5RMP, SEQ ID NOs:32 and 33,respectively), which possesses relatively low activity, by the additionof the 5RMP increased killing such that approximately a 6 log reductionin CFU was obtained, even at the lower concentrations of the peptide (75μg/ml). It was concluded by extrapolation that peptides generated withsequences 120-146WR-5RMP (SEQ ID NO:34) and 120-146WH-5RMP (SEQ IDNO:35) are highly effective, even at substantially lower concentrationsof the peptide.

FIG. 14 shows in detail how the addition of the 5RMP extension (SEQ IDNOs:32-33) to the relatively inactive peptides 120-146QR (SEQ ID NO:30)and 120-146QH (SEQ ID NO:29) enhanced bactericidal activitysignificantly. Peptides 120-146WR (SEQ ID NO:30) and 120-146WH (SEQ IDNO:31) were strongly active in in vitro killing assays, indicating thatthe replacement of the glutamine residue (Q) at position 131 with atryptophan residue (W) markedly increased killing. It can thus bepredicted that a peptide based on 120-146WR-5RMP (SEQ ID NO:34) or120-146WH-5RMP (SEQ ID NO:35) would have superior killing capabilities.

In vivo bactericidal activity of Peptides 120-146QR-5RMP (SEQ ID NO:32),120-146WH (SEQ ID NO:31), and 120-146WR (SEQ ID NO: 30) was alsoinvestigated using the standard Pseudomonas keratitis model in the mouseeye (FIGS. 15-17). FIG. 15 shows that peptide 120-146QR-5RMP (SEQ IDNO:32) significantly reduced the CFU/eye at doses of 10 and 20 mg/ml.Six of the 10 mice in these two groups had no viable bacteria in the eyeafter treatment. FIG. 16 shows that peptide 120-146WH (SEQ ID NO:31)significantly reduced the CFU/eye at doses of 10 and 20 mg/ml. FIG. 17shows that peptide 120-146WR (SEQ ID NO:30) significantly reducedinfection at 5, 10, and 20 mg/ml.

Example 6

Dermal Wound Healing Studies Using 95-122-Based and 120-146-BasedPeptide Compounds

In this Example, peptide compounds of the presently disclosed inventiveconcepts were evaluated as to their activity on the dermal wound healingprocess in a clinically relevant swine full thickness excisional woundmodel. Three different peptide formulations (Group B, Peptide 95-122(SEQ ID NO:26); Group C, Peptide 120-146WH (SEQ ID NO:31); and Group D,Peptide 120-146QH-5RMP (SEQ ID NO:33)) were each evaluated at the sameconcentration (3 mg/ml). Sterile saline served as the control (Group A).

Full thickness excisional wounds were induced on the dorsum of swine(N=3) using a 1.0 cm biopsy punch. Treatments were administered (0.20ml) to each wound (N=6 wounds per group), and the wounds were dressedwith standard occlusive dressing (TEGADERM™, 3M Corporation, St. Paul,Minn.). Treatments were reapplied daily and dressings changed. Clinicalwound healing observations and wound area (2 mm) measurements wereperformed on days 0, 3, 5, 7, 10, and 14 post-wounding. Wound area (2mm) data were expressed as percentage (%) of wound healing. Animals wereterminated on day 14 with wounds harvested and processed forhistological evaluation. Clinical observations revealed no remarkabledifferences between groups for erythema and edema throughout the study.All peptide formulation treatment groups healed faster with greater rateof closure and re-epithelialization over time, as compared to the salinecontrol (Group A).

The results (see FIG. 18 and Table 7) from the quantitative wound areameasurements indicate, in comparison to the saline control Group A, thepercentage (%) of wound healing was greater for Group B (95-122; SEQ IDNO:26) wounds throughout the study. Group B (95-122) demonstrated asignificant (p<0.05) increase in the percentage of wound healing on days5, 7, 10, and 14, with 99.7% of the wounds closed and re-epithelializedby day 10. Groups C (120-146WH, SEQ ID NO:31) and D (120-146QH-5RMP, SEQID NO:33) also demonstrated a significant (p<0.05) increase in thepercentage of wound healing on days 10 and 14 post-wounding. Table 8shows the statistical analysis for the wound healing measurements.

TABLE 7 Swine Wound Healing Results Mean Wound Day: Healing (%) 0 3 5 710 14 Group A- 0.0% 20.1% 30.5% 59.0% 79.2% 88.9% Saline Control GroupB- 0.0% 28.9% 41.5% 70.8% 99.7% 100.0% Peptide Formu- lation 1 (95-122)Group C- 0.0% 22.1% 35.9% 59.7% 93.5% 97.5% Peptide Formu- lation 2(120- 146WH) Group D- 0.0% 22.5% 34.1% 64.1% 93.7% 98.0% Peptide Formu-lation 3 (120- 146QH5RMP)

TABLE 8 Statistical Analysis of Swine Wound Healing Results Day 3 Day 5Day 7 Day 10 Day 14 Group A 0.1407   0.0196 *   0.0350 *  0.0000 *** 0.0000 *** vs Group B Group A 0.7388 0.2264 0.9200 0.0022 ** 0.0030 **vs Group C Group A 0.7032 0.5124 0.4766 0.0041 ** 0.0017 ** vs Group D

FIG. 19 shows representative pictures taken on days 7, 10, and 14demonstrating the extent of wound healing in response to saline (toprow) or peptide 95-122 (SEQ ID NO:26; second row), peptide 120-146WH(SEQ ID NO:31; third row), or peptide 120-146QH-5RMP (SEQ ID NO:33;fourth row). The percent values in Table 7 are measurements taken fromthe results shown in FIG. 19.

The histology correlated with the clinical observations. All peptidetreatment groups (Group B, 95-122 (SEQ ID NO:26); Group C, 120-146WH(SEQ ID NO:31); and Group D, 120-146QH-5RMP (SEQ ID NO:33)) demonstrateda greater extent of epidermal resurfacing and maturation of theepidermis at day 14 post-wounding, as compared to the saline control(Group A). The results from the histological evaluation are provided inFIG. 20 and Table 9. There were minimal differences in the amount orcharacter of granulation tissue, inflammation, and angiogenesis. All thewounds were well filled and vascularized. The major difference was thatGroups B, C, and D demonstrated a greater extent of epidermalresurfacing and maturation of the epidermis (stratification of basal andsuprabasal layers, appearance of a well-organized stratum corneum), ascompared to the saline control.

TABLE 9 Histopathology Results Inflam- Angio- Granu- Epithe- Mean mationgenesis lation lization Group A—Saline Control 1.3 2.3 3.8 1.7 GroupB—Peptide Formulation 1 0.5 2.3 4.0 4.0 (95-122) Group C—PeptideFormulation 2 0.5 1.7 4.0 3.7 (120-146WH) Group D—Peptide Formulation 30.5 1.3 4.0 4.0 (120-146QH5RMP)

Example 7 Treatment of Corneal Wound Healing with CAP37 Protein

Human corneal epithelial cell (HCEC) monolayers were “wounded” thentreated with CAP37, and wound closure was recorded over time. In an invivo model of corneal wound healing, a 2 mm diameter wound was made onthe mouse cornea. Wounds were treated with CAP37, and wound closure wasmonitored at 16 and 24 hours by fluorescein staining. CAP37 treatmentwas shown to facilitate corneal wound healing. Relevant CAP37 sequencesare disclosed in U.S. Pat. No. 7,354,900, which is hereby expresslyincorporated herein by reference in its entirety.

Materials and Methods of Example 7

Cell culture: SV40 adenovirus immortalized HCECs were obtained as agenerous gift from Dr. James Chodosh (Boston, Mass.). HCECs weremaintained in defined keratinocyte-serum free media (KSFM, Gibco, GrandIsland, N.Y.) supplemented with L-glutamine (2 mM, Gibco),antibiotic-antimycotic (0.1 units/mL penicillin G sodium, 100 μg/mLstreptomycin sulfate, 0.25 μg/mL amphotericin B, Gibco), and growthsupplements as provided by the manufacturer. HCECs used in theseexperiments were between passage 10 and 20.

Primary HCECs were isolated from donor corneas acquired from the LionsEye Bank (Oklahoma City, Okla.). Each cornea was quadrisected and placedin Hank's balanced salt solution (HBSS, Gibco) containing dispase (25caseinolytic U/mL; Becton Dickinson Discovery Labware, Bedford, Mass.)and 5 μg/mL gentamicin (A.G. Scientific Inc., San Diego, Calif.). Thecorneal tissue was incubated overnight on ice at 4° C. Cornealepithelial cells were obtained by lifting the epithelial layer from thesurface of the cornea with a scalpel. Following a 5 minute digest in0.25% Trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid, Gibco), anequal amount of heat-inactivated fetal bovine serum (FBS, Gibco) wasadded to the corneal epithelial cells. The cells were centrifuged at450×g for 5 minutes, and the cell pellet was resuspended in KSFMsupplemented with growth factors, as specified by the manufacturer.Cells were seeded and cultured on tissue culture dishes treated with anFNC coating mix containing fibronectin, collagen, and albumin (AthenaES,Baltimore, Md.). Prior to performing all experiments, HCEC cultures wereplaced in KSFM that did not contain growth factors (basal KSFM) for aminimum of 18 hours.

Production of recombinant CAP37: Recombinant CAP37 (rCAP37) was producedin human embryonic kidney (HEK) 293 cells using an RSV-PL4 expressionvector. The recombinant protein was purified on an HPC4 immunoaffinitycolumn as previously described. All preparations of rCAP37 were dialyzedin 0.01% acetic acid and determined to be pure by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.Functional activity was assessed using the modified Boyden chemotaxischamber assay as previously described. rCAP37 preparations used in thesestudies had <0.05 endotoxin units per microgram of protein, asdetermined by the Limulus Amebocyte Lysate assay (QCL 1000, Lonza,Basel, Switzerland).

Animals: C57BL/6 female mice were purchased from Jackson Laboratory (BarHarbor, Me.). Mice were acclimated for 4-7 days and were all 8 weeks ofage at the start of the experiments. The animal research protocols werereviewed and approved by the Institutional Animal Care and Use Committee(IACUC) at the University of Oklahoma Health Sciences Center, OklahomaCity, Okla. and the Dean McGee Eye Institute, Oklahoma City, Okla.

In vitro model of wound healing: An in vitro scratch assay was used inorder to determine the ability of rCAP37 to facilitate corneal woundhealing. Human corneal epithelial cells were cultured as described aboveuntil they reached a confluent monolayer. Each monolayer was scratchedusing a 10 μl pipette tip to create two perpendicular lines. Monolayerswere treated with heparin binding-epidermal growth factor (HB-EGF, 250ng/ml; Becton Dickinson), rCAP37 (25-2000 ng/ml), or basal KSFM (Gibco).Wound closure was monitored at 0, 18, 24, and 48 hours utilizing acamera-equipped inverted microscope (TE2000-E; Nikon, Melville, N.Y.).Time-lapse images of in vitro wound closure were obtained using acamera-equipped inverted microscope (TE2000-E; Nikon) from 0-18 hours.HCEC monolayers were treated with HB-EGF, rCAP37, and basal KSFM asdescribed above. The width of each scratch was quantitated using ImageJsoftware (US National Institutes of Health, Bethesda, Md.). Results arepresented as the percentage of wound closure.

In vivo model of wound healing: Mice were anesthetized using ketamine(100 mg/kg; Bionichepharma, LLC., St. Lake Forrest, Ill.) and xylazine(10 ng/kg; Rompun; Bayer Corp., Shawnee Mission, Kans.), and the rightcornea was wounded as follows. A disposable biopsy punch (2 mm, Miltex,York, Pa.) was used to demarcate the mouse cornea. The cornealepithelium was carefully removed within the 2 mm demarcated area with a0.5 mm burr using the AlgerBrush II (The Alger Company, Inc., LagoVista, Tex.). The corneal abrasions were treated at 0 and 16 hours withHB-EGF (250 ng/ml), rCAP37 (250 ng/ml), or vehicle control (0.9% sodiumchloride, pH 5.5, Baxter, Deerfield, Ill.). Corneal abrasions werevisualized using sterile fluorescein sodium ophthalmic strips USP(FLUORETS®, Chauvin Laboratory, Aubenas, France) dampened with sterilePBS. Images were taken at 0, 16, 24, and 48 hours immediately followingfluorescein staining using a surgical microscope equipped with a camera(Carl Zeiss OPMI VISU 140, Carl Zeiss Surgical, Inc., Oberkochen,Germany). The areas of the open wound were quantitated using ImageJsoftware (US National Institutes of Health), and the results werereported as the percentage of wound closure.

Histology: Whole mouse eyes were collected for histology at 0, 6, 16,24, and 48 hours post wounding and were immediately placed in Preferfixative (Anatech LTD., Battle Creek, Mich.) for 20 minutes before beingtransferred to 70% ethanol. Tissues were paraffin-embedded and cut at athickness of 5 μm, mounted on SUPERFROSTPLUS® slides (Statlab MedicalProducts, Lewisville, Tex.), and subsequently deparaffinized,rehydrated, and washed in deionized water. Sections were stained withhematoxylin (Leica Microsystems, Buffalo Grove, Ill.) and rinsed twicein deionized water before being washed in Blue Buffer (LeicaMicrosystems). The sections were finally washed in deionized water and95% ethanol prior to being counterstained with eosin (LeicaMicrosystems). Sections were dehydrated in ethanol and cleared inxylene.

Immunohistochemistry: Whole mouse eyes were collected, fixed, embedded,and cut as previously described. Antigen retrieval was performed bytreating sections with Rodent Decloaker (BioCare Medical, Concord,Calif.) and steaming them for 20 minutes before cooling with deionizedwater for 20 minutes. Sections were blocked for 30 minutes in RodentBlocker M (BioCare Medical, Concord, Calif.), washed three times for 5minutes per wash in deionized water, and blocked in peroxide block (CellMarque, Rocklin, Calif.) for 10 minutes. Sections were then washed threetimes for 5 minutes in deionized water and incubated overnight at 4° C.with rabbit anti-PKCδ (4 μg/ml; Santa Cruz Biotechnology, Inc. SantaCruz, Calif.). Sections incubated with rabbit IgG (Cell SignalingTechnology, Danvers, Mass.) overnight at 4° C. served as controls fornonspecific staining. After incubation with primary antibody, sectionswere washed three times for 5 minutes in Tris-buffered saline (TBS) andincubated with rabbit-on-rodent horseradish peroxidase (HRP)-polymer(BioCare Medical) for 30 minutes. Following three, 5 minute washes inTBS, sections were stained with 3′,3′-diaminobenzidinetetrahydrochloride (DAB) chromogen (Cell Marque, Rocklin, Calif.),washed in deionized water, and counterstained with Immuno MasterHematoxylin (American Master*Tech Scientific, Inc., Lodi, Calif.).Images of the stained tissues were obtained using an inverted microscope(TE2000-E; Nikon) equipped with a camera.

Immunofluorescence: HCECs and primary HCECs were cultured on Lab-Tek® IIglass chamber slides (Nunc, Rochester, N.Y.) and starved overnight inbasal media. For wounding studies, monolayers were scratched with a 10μl pipette tip or left unscratched and treated with 1 μM phorbol12-myristate 13-acetate (PMA, Sigma-Aldrich), rCAP37 (25-500 ng/ml), orbasal KSFM (Gibco) for 15 minutes. After treatment, the cells were fixedin 4% (v/v) formaldehyde solution (Thermo, Rockford, Ill.) in PBS(Gibco) for 20 minutes at room temperature followed by permeabilizationin 0.5% TRITON™-X 100 (Mallinckrodt, St. Louis, Mo.) in PBS for 10minutes. Remaining formaldehyde was quenched with 0.05 M ammoniumchloride (NH₄CI, Sigma-Aldrich, St. Louis, Mo.) in PBS for 10 minutes.Cells were washed in PBS and incubated in blocking buffer (10% (v/v)normal goat serum in PBS containing 5% bovine serum albumin (BSA,Calbiochem, Gibbstown, N.J.)) and 0.5% TRITON™-X 100 (Mallinckrodt, St.Louis, Mo.) at room temperature for 1 hour. To detect the PKC isoforms,cells were incubated in primary mouse antibodies (Becton DickinsonDiscovery Labware) directed against PKCδ (250 ng/ml), PKCθ (500 ng/ml),PKCα (1 μg/ml), or PKCγ (1 μg/ml) for 1 hour at room temperature. MouseIgG (1 μg/ml; Jackson ImmunoResearch) served as the control fornonspecific staining. The cells were washed in PBS containing 0.25%TRITON™-X 100 (Mallinckrodt) and incubated in secondary antibody (4μg/ml in blocking buffer; ALEXA FLUOR® 488 dye (Life Technologies Corp.,Grand Island, N.Y.)) for 1 hour at room temperature. Cells were washedthree times for 5 minutes in PBS followed by a final water wash andmounted using PROLONG® Gold Antifade containing DAPI (MolecularProbes/Life Technologies Corp., Grand Island, N.Y.). Images wereobtained using an inverted epifluorescent microscope (TE2000-E; Nikon).

siRNA transfection and gene silencing: Stealth RNAi™ (10 μM, Ambion®,Grand Island, N.Y.) directed against PKCδ or Stealth RNAi™ siRNANegative Control Hi GC (10 μM, Ambion®) was delivered into the mouseconjunctiva through a 5 μl subconjunctival injection using a 33-gaugeneedle (Hamilton®, Reno, Nev.). After subconjunctival injection, thecorneas were immediately wounded using the AlgerBrush II as describedabove. Wound closure was quantitated at 16 and 24 hours as described.Animals were humanely euthanized at 24 hours, and each cornea wasexcised using a SklarSafe™ Safety Scalpel #11 (SKLAR®, West Chester,Pa.). Tissues were immediately flash frozen. Corneal homogenates wereprepared as described below and analyzed for levels of PKCδ by Westernblot analysis to confirm the efficiency of each knockdown. The level ofPKCδ in the knockdown cornea was compared to the Stealth RNAi™ siRNANegative Control Hi GC (Ambion®).

Protein extraction and Western blotting: Mouse corneas were excisedusing a SklarSafe™ Safety Scalpel #11 (SKLAR®) and frozen at theindicated time points as described above. Corneas were placed in 200 μlof radioimmunoprecipitation assay (RIPA) buffer containing a 1× cocktailof complete ULTRA Protease Inhibitors (Roche Diagnostics Corp.,Indianapolis, Ind.). Tissue homogenates were created by disrupting thecorneas for 10 minutes at maximum speed in the BULLET BLENDER® (NextAdvance, Inc., Averill Park, N.Y.) using 0.9-2 mm stainless steel beads(Next Advance, Inc.). The homogenates were centrifuged at 16,000×g for10 minutes, and the protein concentration of the supernatant from eachcorneal homogenate sample was determined using a BCA proteinconcentration assay (Pierce, Rockford, Ill.).

Protein (20 μg) from each sample of corneal homogenate was analyzed byelectrophoresis on a 10% SDS-PAGE gel. Following electrophoresis,samples were transferred to nitrocellulose membranes (Whatman® Inc.Florham Park, N.J.) for Western blot analysis. Nitrocellulose membranes(Whatman® Inc.) were blocked for 1 hour in 5% BSA (Calbiochem) in TrisBuffered Saline with TWEEN®20 (Thermo Fisher Scientific, Pittsburgh,Pa.) (TBST) and then incubated overnight at 4° C. with 5% BSA(Calbiochem) in TBST containing primary antibodies directed against PKCδ(Santa Cruz) or (3-actin (Sigma-Aldrich) as specified by themanufacturers. The membranes were washed three times for 5 minutes inTBST before being incubated at room temperature for 1 hour with rabbit(Cell Signaling Technology, Danvers, Mass.) or mouse (Sigma-Aldrich)secondary antibody conjugated to HRP. Secondary antibodies were used asdirected by the manufacturer. Blots were developed using Pierce ECLWestern Blotting Substrate and visualized using the UltraLum Imager(Omega, Claremont, Calif.). Blots were analyzed and semi-quantitatedusing ImageJ software (U.S. National Institutes of Health, Bethesda,Md.).

Statistics: In vitro wound healing experiments were analyzed using aone-way analysis of variance (ANOVA) followed by a Dunnett's multiplecomparison test. In vivo wound healing and PKCδ knockdown studies wereanalyzed using an unpaired t-test. GraphPad Prism 4.03 (GraphPadSoftware, Inc., San Diego, Calif.) was used for statistical analysis.The independent means of experimental values are shown±SEM. P<0.05 wasconsidered significant for all statistical analyses.

Results of Example 7

CAP37 facilitates wound closure in vitro: Previous studies have shownthat CAP37 mediates HCEC proliferation, migration, and adhesion, leadingthe inventors to hypothesize that CAP37 may facilitate the process ofcorneal wound healing. To investigate this premise, an in vitro scratchmodel was utilized to determine if CAP37 could promote wound closure.The findings show that CAP37 promoted wound closure in vitro in adose-dependent manner (FIG. 21A). CAP37 maximally facilitated woundclosure when used at concentrations between 250 and 500 ng/ml. Thepercentage wound closure in CAP37-treated wounds with 250 ng/ml of CAP37was almost 71% at 18 hours and was significantly greater (**P<0.01) thanbasal media-treated samples that showed approximately 41% closure.Treatment of wounds with 500 ng/ml of CAP37 resulted in approximately62% closure and was significantly greater than the buffer control(*P<0.05). HB-EGF, which was used as the positive control, showed almost88% closure of treated monolayers (FIG. 21A). Representative images ofthe in vitro scratch assay taken at each time point show the extent ofclosure in response to the various treatments. Wounds treated withHB-EGF were completely closed by 24 hours, whereas wounds treated with250 ng/ml of CAP37 required between 24 to 48 hours for complete closureto occur. Buffer-treated wounds did not reach full closure at 48 hoursof treatment (FIG. 21B).

Time-lapse microscopy studies of in vitro wound closure during the first18 hours post wounding revealed differences in the manner in which theleading edge of cells responded to HB-EGF versus CAP37. In theCAP37-treated samples, it was noticed that individual cells would detachfrom the leading edge and crawl rapidly across the wounded areaindependent of the other cells. The cells showed polarization, withobvious lamellipodia indicative of activation. Not all cells at theleading edge showed activation or produced lamellipodia. However, theedge of the wound displayed more dynamic activity than the HB-EGFtreated monolayers. HB-EGF-treated cells showed a strikingly differentmethod of wound closure. These cells appeared to advance in a sheet, andindividual cells did not detach or appear to migrate independently ofthe advancing sheet of cells across the wound. Morphological changes inthe CAP37-treated cells, such as polarization and lamellipodiaformation, were not as apparent in the HB-EGF or basal KSFM treatedmonolayers.

CAP37 facilitates corneal wound healing in an In Vivo Mouse Model: Usingthe in vivo mouse model of corneal wound healing described in thematerials and methods section, the effect of topical application ofCAP37 on wound closure was explored, and the rate of closure at 16, 24,and 48 hours was compared with the positive control HB-EGF and thenegative vehicle control (FIGS. 22A and 22B). At 16 hours, CAP37 hadeffectively reduced the size of the wound by 71%, and by 24 hours theCAP37-treated (250 ng/ml) wounds were 83% closed. At both time points,the CAP37 treated wounds were closed significantly more than the vehicletreated controls (*P<0.05 compared to the buffer control). A timedependent closure of wounds was also demonstrated with HB-EGF and wassignificantly higher compared to the buffer control (**P<0.01).Representative images of fluorescein stained wounds are shown in FIG.22B and demonstrate the time-dependent healing of the wounds in responseto treatment with vehicle, HB-EGF and CAP37. As can be seen, all woundsindicated complete closure by 48 hours, as measured by fluoresceinstaining (FIG. 22B).

CAP37 leads to corneal re-epithelialization by 24 Hours: The fluoresceinstaining method provides a gross morphologic approach to determine theextent of corneal abrasion and healing. However, to determine whetherCAP37 promotes complete re-epithelialization and restores the structuralintegrity of the epithelial layers of the cornea, whole eye globes werecollected at 16, 24, and 48 hours and processed for histology (FIG.23A-E). Hematoxylin and eosin (H&E) stained sections revealed thatre-epithelialization was well underway by 24 hours with restoration ofthe basal cell layers of the epithelium, indicating the proliferation ofthese cells in response to treatment with CAP37 (FIG. 23A). Themigration and differentiation to squamous cells at 24 hours in responseto CAP37 did not appear to be complete, possibly accounting for thedetection of the low level of fluorescein staining at 24 hours shown inFIG. 22B. The re-epithelialization in response to CAP37 was greatlyaccelerated in comparison to the vehicle-treated wounds at 24 hours(FIG. 23B). As can be seen, the epithelium was only a single-layer thickin the central region of the wound, and proliferation of the basal cellswas limited in comparison to the CAP37-treated wounds at this same timepoint. This is further confirmed in the fluorescein-stained wounds inFIG. 22B, showing that the vehicle-treated wounds had much strongerstaining. At 48 hours, the CAP37-treated wounds (FIG. 23C) had regainedfull integrity and could not be histologically differentiated from theunwounded corneas (FIG. 23E). Although the vehicle-treated cornea hadre-epithelialized by 48 hours, complete structural integrity of theapical layers was not observed (FIG. 23D).

PKCδ and PKCθ are Present in Wounded HCEC Monolayers: Studies by theinventors show that PKC isoforms α, δ, ε, θ, η, ι, λ, and ζ areexpressed in human corneal epithelial cells and that CAP37 specificallyactivates PKC δ and θ during chemotaxis. Since migration of epithelialcells is an important step in normal wound healing, it was questionedwhether these isoforms were involved in wound healing. Unscratched andscratched HCEC monolayers were stained for the expression of PKC δ andθ. Results showed the constitutive expression of PKC isoforms δ and θ inunscratched HCEC monolayers and demonstrated the increased staining ofboth isoforms along the wound edge (FIG. 24A). The constitutiveexpression of PKC isoforms δ and θ was confirmed using primary HCECs(FIG. 24B). The specificity of the staining for these two isoforms inthe SV40 HCEC cell line and primary HCECs was demonstrated using an IgGantibody control which showed no staining.

CAP37 treatment leads to an increase in PKCδ Staining in HCECMonolayers: PKCδ was selected for further investigation inCAP37-mediated corneal wound healing. To determine if CAP37 had aneffect on PKCδ expression in HCEC monolayers that were unscratched andscratched, HCECs were treated with CAP37 (250 and 500 ng/ml) and apositive control, PMA. A dose-dependent increase in PKCδ staining wasseen at 15 minutes in CAP37-treated cells in both non-scratched (FIGS.25E and 25G) and scratched (FIGS. 25F and 25H) monolayers over untreatednon-scratched (FIG. 25A) and scratched (FIG. 25B) monolayers. Theincrease in PKCδ staining appeared to be dose-dependent, with greaterstaining observed following treatment with 500 ng/ml of CAP37. Theincrease in PKCδ staining in response to CAP37 was sustained for atleast 18 hours (FIG. 25L) versus the untreated control (FIG. 25K). Theexpression of PKCδ was comparable between CAP37-treated cells (FIG.25E-H) and PMA-treated cells (FIG. 25C, 25D). To establish that thisincreased expression in response to CAP37 treatment was specific forPKCδ, staining for the a isoform of PKC was also performed, but noincrease in PKCα staining was found either within the monolayer (FIG.251) or along the wound edge (FIG. 25J) of the monolayer when treatedwith CAP37.

PKCδ is Expressed Along the Wound Edge In Vivo: Since staining for PKCδwas increased in response to wounding in cultured HCEC monolayers (FIG.24A), studies were performed to determine whether an increase inexpression of PKCδ would be seen in corneal wounds in vivo. Corneas wereabraded as described previously, and the whole mouse eye globes werecollected at 6, 16, and 48 hours post wounding for immunohistochemistryin order to determine the expression of PKCδ in response to wounding.Little to no detection of PKCδ was observed at the leading edge of thenewly proliferating and migrating epithelial cells at 6 hours (FIG.26A-B) and 16 hours (FIG. 26C) post wounding. However, the epithelialcells at a distance from the leading edge showed a low level of stainingthat was comparable to the constitutive expression of PKCδ in normalunwounded cornea (FIG. 26G).

To determine if CAP37 treatment of the wounds had an effect on PKCδexpression, corneas were wounded and treated immediately and 16 hoursfollowing wounding. Eyes that were enucleated at 6 and 16 hours had onetreatment of CAP37 (0 hours) whereas the eyes that were enucleated at 48hours were treated twice with CAP37 (0 and 16 hours). Sections stainedat 6 hours showed strong staining for PKCδ in the newly migrating andproliferating epithelial cells as well as those cells distant from thewound edge (FIG. 26D-E). A similar staining pattern for PKCδ was seen insections that were obtained at 16 hours post wounding (FIG. 26F).Sections that were obtained at 48 hours were completely healed andshowed uniform staining throughout the epithelium (FIG. 26H) and was atan intensity similar to constitutive expression in unwounded corneas(FIG. 26G).

PKCδ is necessary for CAP37 wound healing in vivo: In vivo experimentswere performed using siRNA to determine if CAP37 mediates wound healingvia PKCδ. Mouse corneas were transfected with siRNA directed againstPKCδ or with scrambled siRNA. Wounds were created as described followingtransfection with scrambled siRNA and PKCδ siRNA. Corneas were thentreated with the vehicle or CAP37 (250 ng/ml), and wound closure wasmeasured at 16 and 24 hours. Corneas transfected with scrambled siRNAshowed the expected increase in wound closure following CAP37 treatmentat 24 hours (P<0.05) (FIG. 27A). In corneas transfected with PKCδ siRNA,there was no significant increase in wound healing in response to CAP37(FIG. 27A) at either the 16 or 24 hour time points. CAP37 treatment didlead, however, to a slight increase in wound healing over saline treatedwounds, but this increase did not reach statistical significance (FIG.27A). This could indicate that PKCδ is not the only signaling moleculeinvolved, but is more likely a reflection that the knockdown of PKCδ ineach cornea, as assessed by Western blot, was on average 50% (FIG. 27A).The level of knockdown was determined to be statistically significant(*** P<0.005) when compared with the scrambled siRNA controls.Representative images of fluorescein stained wounds depict the extent ofwound closure over time in corneas transfected with scrambled siRNA andtreated with CAP37 (FIG. 27B). As can be seen, maximum wound closure wasobserved in animals that were transfected with scrambled siRNA andtreated with CAP37.

These results demonstrate a novel function for the neutrophil-derivedprotein CAP37, also known as heparin binding protein and azurocidin.Using a series of in vitro and in vivo models of wound healing, it wasshown that CAP37 accelerates wound closure in vitro as well as in amouse model of corneal abrasion. Importantly, the mechanism wherebyCAP37 facilitates corneal epithelial wound healing has been identified.By employing immunohistochemical and siRNA techniques, it wasestablished that the protein kinase C (PKC) signaling pathway,specifically PKCδ, is the key modulator of CAP37-mediated cornealepithelial wound healing. This appears to be the first demonstration ofthe intracellular signaling mechanism employed by a neutrophilgranule-derived antimicrobial protein in corneal epithelial woundhealing.

The cornea is an immune privileged site, and therefore the process ofhealing in the cornea is not identical to the process that occurs indermal skin wounds. However, one key feature in both corneal and dermalwound healing is that neutrophils are an essential cellular component.Neutrophils are early participants in the process and are fundamental toprotecting the host from infection due to their potent antimicrobial andphagocytic activity. When the cornea is injured, neutrophils migratethrough the limbal vessels into the cornea. Studies have shown thatdelayed corneal wound healing occurs in mice with antibody-inducedneutropenia. Other studies using wild type and knockout mice for lumicanand heme oxygenase, and rabbit models of corneal epithelial woundhealing, have further established that the presence of neutrophilsaccelerates healing. This led the inventors to the concept thatantimicrobial proteins found within the granules of neutrophils such asCAP37, LL-37, human β-defensin-1 (HBD-1), andbactericidal-permeability-increasing (BPI) protein may prove to beuseful in modulating wound closure.

Neutrophils that are recruited to the wound site release their granulecontents, including CAP37 and other antimicrobial proteins and peptides,which provide the first line of defense against corneal infection. It isnow known that these antimicrobial peptides, in addition to killing theinvading pathogens, are able to modulate functions of host cells thatregulate innate immunity. Importantly, the neutrophil is not the onlysource of these antimicrobial proteins. CAP37 and LL-37 can be inducedin host cells, including the corneal epithelium, in response toinfection and wounding. LL-37 has been shown, like CAP37, to beantimicrobial, bind lipopolysaccharide, and promote corneal epithelialwound healing in vitro. Unlike CAP37, LL-37 does not promote HCECproliferation, and the intracellular signaling mechanisms involved inits effects on corneal epithelial cell migration and wound healing havenot been elucidated.

This Example not only confirmed that HB-EGF promotes wound healing invitro, but also revealed for the first time that HB-EGF promotes cornealwound healing in vivo. HB-EGF was selected for use as a positivecontrol, as it is also a heparin binding protein like CAP37 and becausethere is in vitro evidence that HB-EGF facilitates corneal wound healingin organ tissue cultures. Previous in vitro studies have indicated thatHB-EGF, but not EGF, facilitates corneal wound healing through theprolonged activation of the EGF receptor. This is believed to be due tothe fact that HB-EGF is able to bind to the negatively charged glycanson the corneal surface, while EGF is washed away after treatment. Theheparin binding characteristics of HB-EGF that make it desirable as along acting alternative to EGF also apply to CAP37, making it aneffective therapeutic for ocular wound healing.

Without wishing to be bound by theory, the present work indicates thatthe CAP37-mediated cellular processes, previously defined asproliferation, migration, and adhesion, are working in conjunction tofacilitate corneal epithelial wound healing. Time-lapse videos ofCAP37-treated in vitro wounds infer that CAP37 affects cornealepithelial wound healing by primarily facilitating migration, at leastin the early stages of wound repair. Other mechanisms, such asproliferation and adhesion, may be involved at later stages of theprocess.

One component of the work was the delineation of the intracellularsignaling mechanism that evoked CAP37-mediated corneal epithelial woundhealing. After demonstrating the ability of CAP37 to mediate cornealwound closure in vitro and in vivo (FIGS. 21-22), the presence of PKCδwas identified in untreated corneal epithelial cell culture monolayersand confirmed in primary corneal epithelial cells (FIG. 24).CAP37-treated corneal epithelial cell monolayers showed an increase instaining for PKCδ (FIG. 25) that persisted up to 18 hours after CAP37treatment, indicating that the effect was not transient. Whileimmunohistochemistry revealed the constitutive presence of PKCδ inunwounded mouse corneas, immunohistochemistry of CAP37-treated woundsrevealed an increase in PKCδ staining along the leading edge of thewound at both 6 hours and 16 hours compared to vehicle-treated controls(FIG. 26). The presence of PKCδ along the wound edge and an increase inPKCδ in CAP37-treated wounds prompted studies in which PKCδ was knockeddown in a mouse model of corneal epithelial wound healing. Resultsrevealed that a partial knockdown of PKCδ is sufficient to reduce theeffect of CAP37 on corneal wound healing in vivo. While the PKCδknockdown did not entirely ablate the effects of CAP37, and there was nosignificant difference between the CAP37 treated wounds transfected withscrambled or PKCδ siRNA at either time point, the decrease in woundhealing may still have a significant impact clinically (FIG. 27). Theaverage PKCδ knockdown achieved was 50% and could explain why CAP37still promoted a certain amount of wound closure in corneas transfectedwith siRNA directed against PKCδ. Another explanation for these findingsis that other PKC isoforms such as PKCθ may partially contribute toCAP37-mediated wound healing. Others have shown that PKCα and PKCε areimportant in HGF-induced corneal wound healing, and that PKCα is a keymodulator in rabbit corneal wound healing. In contrast, studies usingPKCα knockout mice have demonstrated more rapid corneal epithelial woundhealing, and the inventors indicate that this is perhaps due to fewerinfiltrating neutrophils in this model. The studies with CAP37 did notshow the involvement of PKCα. As with all inflammatory reactions, a finebalance exists between limiting the influx of inflammatory cells andpromoting the healing process.

An interesting observation in FIG. 26 was the staining of the emigratingleukocytes in addition to the staining of PKCδ at the leading edge ofCAP37 treated corneal wounds. Neutrophils are known to express PKCδ,which is required for the full NADPH oxidation and respiratory burstactivation in neutrophils. Studies have shown that antimicrobialproteins such as LL-37 can induce the production of reactive oxygenspecies (ROS) in neutrophils in a time- and dose-dependent mannerthrough the NADPH oxidase system. This is of particular relevance interms of wound healing, because low levels (10-20 μM) of hydrogenperoxide (H₂O₂) have been shown to induce corneal wound healing throughthe promotion of adhesion and migration of corneal epithelial cells.Unpublished results from the inventors showed that CAP37 increases theproduction of ROS in microglia and monocytes, cells in which CAP37 alsomediates chemotaxis. It was also recently demonstrated that the NADPHoxidase is expressed in HCECs, and that these cells are also capable ofproducing superoxide through this enzymatic complex. Taken together,these studies indicate that CAP37 induces the expression of PKCδ in thecorneal epithelium cells at the edge of the wound, thereby locallyactivating the NADPH oxidase and the production of ROS to facilitatewound healing.

Example 8

Synergism Between BCC02-5RMP (SEQ ID NO:21) and Low Dosage Antibiotics

Traditional efforts to counter antibiotic resistance have relied uponmaking incremental changes to existing drugs. This strategy providesshort-term relief, but bacteria quickly develop resistance to theseslight modifications. A serious gap exists in the drug developmentpipeline for safe and effective therapies for treating infections due tomultidrug resistant Gram negative organisms. As discussed elsewhereherein, the peptide compounds of the presently disclosed inventiveconcepts contributes to the alleviation of this major healthcare problemas a new class of anti-infectives for the treatment of seriousinfections due to Gram negative pathogens, such as but not limited to,Pseudomonas sp., Acinetobacter sp., Salmonella sp., and E. coli. Thepresently disclosed inventive concepts thus include as embodiments amethod of enhancing the efficacy of an antibiotic in the treatment of abacterial infection. In the method, the antibiotic and the presentlydisclosed peptide compound are administered; the antibiotic isadministered in an amount which (i) has suboptimal activity or isineffective against the bacteria when administered alone, and (ii) iseffective against the bacteria when administered in combination with thepeptide compound.

The present therapeutic treatment uses, in at least one embodiment, thepresently disclosed peptide compounds (e.g., BCC02-5RMP (SEQ ID NO:21))to potentiate (enhance) the effect of standard of care antibiotics,making these antibiotics effective against resistant bacteria at dosagelevels previously considered to be suboptimal or ineffective. Thesefindings are significant because, if existing antibiotics can be used ata lower dose (suboptimal or <MIC), they are less likely to be toxic; inaddition, when an existing antibiotic is administered at a lower dose(suboptimal or <MIC) and in combination with a peptide compound of thepresently disclosed inventive concepts, bacteria are less likely todevelop resistance to the antibiotic as compared to when the lower doseof antibiotic is administered on its own. Thus, administration of anantibiotic in combination with a peptide compound of the presentlydisclosed inventive concepts is more likely to extend the length of timethe antibiotic can be used in the clinical setting.

The importance of these findings as explained above is that thepresently disclosed peptide compounds, such as but not limited toBCC02-5RMP (SEQ ID NO:21), can potentiate the effect of standard of careantibiotics such that they can be used at a lower dose (suboptimal or<MIC) and regain their activity against organisms that have becomeresistant to it. In other words, the organism can be re-sensitized toantibiotics that were once thought to be ineffective against theorganism due to resistance thereto.

Data in support of this result are shown in FIGS. 28-30. A clinicalisolate of P. aeruginosa that was resistant to Levofloxacin andCiprofloxacin with intermediate sensitivity to Cefotaxime was selected.The peptide and antibiotics were set up in the wells of the microtiterplate at starting ratios of MIC equivalents of antibiotic:peptide at3:1, 1:1, and 1:3. They were serially diluted to concentrations wellbelow suboptimal/sublethal levels of the antibiotic and peptide. Theorganism was added (1×10⁵ CFU/well), the BIOSCREEN C™ assay (GrowthSystems USA, Piscataway, N.J.) was performed, and data was collectedover 24 hours. The results showed that although this organism wasresistant to Ciprofloxacin (MIC of 8.4 μg/ml), Levofloxacin (MIC of 15μg/ml), and Cefotaxime (MIC of 15 μg/ml), it was possible to make thePseudomonas isolate sensitive to each of these antibiotics at asuboptimal/sublethal dose with the addition of the peptide.

For example, FIG. 28A shows the growth/survival curve of the organism inthe presence of Cefotaxime at a sublethal concentration of 2.81 μg/ml,and growth curves with combinations of Cefotaxime at 2.81 μg/ml andpeptide at 0.34, 1.01, and 3.04 μg/ml. The growth of the organism in thepresence of peptide on its own at 3.15 μg/ml is shown. The growth of thePseudomonas in the absence of peptide and antibiotic is also shown. Aswill be seen from the growth curve as well as the histogram showingfractional area (FA) (FIG. 28B), the addition of 2.8 μg/ml of Cefotaximehad very little bactericidal impact on the organisms, as growth in thepresence of the antibiotic was very similar to the control growth curve.The addition of 0.34 and 1.0 μg/ml of peptide BCC02-5RMP (SEQ ID NO:21)had no statistical effect on the killing effect of the antibiotic.However, on the addition of 3.04 μg/ml of peptide, it was apparent thatthe bacterium with intermediate sensitivity to Cefotaxime was nowsensitive (P=0.0143 by unpaired t test). This shows that whenCAP37-based peptide compounds were used in combination with standardantibiotics, the therapeutic dose of the standard antibiotic waslowered, thus making a resistant organism sensitive to therapy.

To establish whether this effect might be seen with antibiotics from adifferent class, it was selected to perform these studies withCiprofloxacin (FIG. 29A-B) and Levofloxacin (FIG. 30A-B). The additionof 4.05 μg/ml of peptide to 2.1 μg/ml of Ciprofloxacin significantlyaffected killing (P=0.0003 by unpaired t test). In other words, themethods described herein were able to reduce the MIC from 8.4 μg/ml to2.1 μg/ml (FIG. 29A-B). Similarly, the methods were able to reduce theMIC of Levofloxacin from 15 μg/ml to 3.5 μg/ml in the presence of 3.04μg/ml of the peptide (FIG. 30A-B).

While the presently disclosed inventive concepts have been describedherein in connection with certain embodiments so that aspects thereofmay be more fully understood and appreciated, it is not intended thatthe presently disclosed inventive concepts be limited to theseparticular embodiments. On the contrary, it is intended that allalternatives, modifications and equivalents are included within thescope of the presently disclosed inventive concepts as defined herein.Thus the examples described above, which include particular embodiments,will serve to illustrate the practice of the presently disclosedinventive concepts, it being understood that the particulars shown areby way of example and for purposes of illustrative discussion ofparticular embodiments of the presently disclosed inventive conceptsonly and are presented in the cause of providing what is believed to bethe most useful and readily understood description of procedures as wellas of the principles and conceptual aspects of the inventive concepts.Changes may be made in the formulation of the various compositionsdescribed herein, the methods described herein or in the steps or thesequence of steps of the methods described herein without departing fromthe spirit and scope of the presently disclosed inventive concepts.

What is claimed is:
 1. A composition comprising a peptide compoundrepresented by of Formula (I):S—X_(R)-Pep-Y_(R) wherein: Pep is one of SEQ ID NOs:30 and 31; X_(R) andY_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginine residues,with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; andS is a solubilizing moiety comprising 1-10 subunits having the formula:NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO— wherein q=0, 1, 2, 3, or 4;p=1, 2, 3, 4, 5, 6, 7, or 8; R₁ and R₂ are selected from the groupconsisting of H, CH₃, and CH₂CH₃; n=1 or 2; and with the proviso that Scomprises at least one subunit which is not NH₂CH₂CH₂OCH₂CH₂OCH₂CO—(AEEA) or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— (AEEEA).
 2. The composition ofclaim 1, wherein X_(R) and Y_(R) are selected from the group of (X_(R),Y_(R)) pairs consisting of (5,0), (4,1), (3,2), (2,3), (1,4), and (0,5).3. The composition of claim 1, wherein the peptide compound is furtherdefined as comprising the sequence of one of SEQ ID NOs:34and
 35. 4. Thecomposition of claim 1, further comprising at least one additionaltherapeutic agent selected from the group consisting of antibacterialagents and antifungal agents.
 5. A method of treating and/or inhibitingat least one of a bacterial infection and a fungal infection in asubject in need of such treatment, the method comprising the step of:administering to the subject a composition comprising a peptide compoundof Formula (I):S—X_(R)-Pep-Y_(R) wherein: Pep is one of SEQ ID NOs:30 and 31; X_(R) andY_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginine residues,with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; andS is a solubilizing moiety comprising 1-10 subunits having the formula:NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO— wherein q=0, 1, 2, 3, or 4;p=1, 2, 3, 4, 5, 6, 7, or 8; R₁ and R₂ are selected from the groupconsisting of H, CH₃, and CH₂CH₃; n=1 or 2; and with the proviso that Scomprises at least one subunit which is not NH₂CH₂CH₂OCH₂CH₂OCH₂CO—(AEEA) or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— (AEEEA).
 6. The method of claim5, wherein: (a) the bacterial infection is caused by a Gram negativebacterium selected from the group consisting of Pseudomonas aeruginosa,Escherichia coli, Salmonella typhimurium, and Acinetobacter baumannii;and/or (b) the fungal infection is caused by a fungal organism selectedfrom the group consisting of Candida spp., Saccharomyces cerevisiae,Histoplasma spp., Histoplasma capsulatum, Aspergillus spp., Aspergillusfumigatus, and Cryptococcus neoformans.
 7. The method of claim 5,wherein the composition further comprises at least one additionaltherapeutic agent selected from the group consisting of antibacterialagents and antifungal agents.
 8. The method of claim 5, furthercomprising the step of administering to the subject at least oneadditional therapeutic agent simultaneously or wholly or partiallysequentially with the composition.
 9. A method of treating at least oneof a wound and a graft to promote healing of the wound and/or acceptanceof the graft in a subject in need of such treatment, the methodcomprising the step of: administering to the subject a compositioncomprising a peptide compound of Formula (I):S—X_(R)-Pep-Y_(R) wherein: Pep is one of SEQ ID NOs:30 and 31; X_(R) andY_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginine residues,with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; andS is a solubilizing moiety comprising 1-10 subunits having the formula:NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO— wherein q=0, 1, 2, 3, or 4;p=1, 2, 3, 4, 5, 6, 7, or 8; R₁ and R₂ are selected from the groupconsisting of H, CH₃, and CH₂CH₃; n=1 or 2; and with the proviso that Scomprises at least one subunit which is not NH₂CH₂CH₂OCH₂CH₂OCH₂CO—(AEEA) or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— (AEEEA).
 10. The method ofclaim 9, wherein: (a) the wound is selected from the group consisting ofa surface wound, a laceration, an abrasion, an avulsion, an incision, anamputation wound, a diabetic ulcer of the leg and/or foot, a pressuresore, a bed sore, a wound due to peripheral vascular disease, a burn, apost-surgical wound, an ocular ulcer, dry eye, an ocular wound, andcombinations thereof; and/or (b) the graft is a skin graft.
 11. Themethod of claim 9, wherein the composition further comprises at leastone additional therapeutic agent selected from the group consisting ofantibacterial agents and antifungal agents.
 12. The method of claim 9,further comprising the step of administering to the subject at least oneadditional therapeutic agent simultaneously or wholly or partiallysequentially with the composition.
 13. A method of enhancing theefficacy of an antibiotic in the treatment of a bacterial infection, themethod comprising the steps of: administering to the subject acomposition comprising a peptide compound of Formula (I):S—X_(R)-Pep-Y_(R) wherein: Pep is one of SEQ ID NOs:30 and 31; X_(R) andY_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginine residues,with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; andS is a solubilizing moiety comprising 1-10 subunits having the formula:NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO— wherein q=0, 1, 2, 3, or 4;p=1, 2, 3, 4, 5, 6, 7, or 8; R₁ and R₂ are selected from the groupconsisting of H, CH₃, and CH₂CH₃; n=1 or 2; and with the proviso that Scomprises at least one subunit which is not NH₂CH₂CH₂OCH₂CH₂OCH₂CO—(AEEA) or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— (AEEEA); administering to thesubject an antibiotic.
 14. The method of claim 13, wherein theantibiotic is administered simultaneously or wholly or partiallysequentially with the composition.
 15. A method of enhancing theefficacy of an antibiotic in the treatment of at least one of a woundand a graft to promote healing of the wound and/or acceptance of thegraft in a subject in need of such treatment, the method comprising thesteps of: administering to the subject a composition comprising apeptide compound of Formula (I):S—X_(R)-Pep-Y_(R) wherein: Pep is one of SEQ ID NOs:30 and 31; X_(R) andY_(R) are each independently 0, 1, 2, 3, 4, 5, or 6 arginine residues,with the proviso that (X_(R)+Y_(R)) is 4, 5, or 6 arginine residues; andS is a solubilizing moiety comprising 1-10 subunits having the formula:NH_(n)CH₂CH₂—(OCH₂CH₂)_(p)—O(CR₁R₂)_(q)CO— wherein q=0, 1, 2, 3, or 4;p=1, 2, 3, 4, 5, 6, 7, or 8; R₁ and R₂ are selected from the groupconsisting of H, CH₃, and CH₂CH₃; n=1 or 2; and with the proviso that Scomprises at least one subunit which is not NH₂CH₂CH₂OCH₂CH₂OCH₂CO—(AEEA) Or NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CO— (AEEEA); administering to thesubject an antibiotic.
 16. The method of claim 15, wherein theantibiotic is administered simultaneously or sequentially with thecomposition.
 17. The method of claim 5, wherein X_(R) and Y_(R) areselected from the group of (X_(R), Y_(R)) pairs consisting of (5,0),(4,1), (3,2), (2,3), (1,4), and (0,5).
 18. The composition of claim 1,further comprising: a pharmaceutically-acceptable carrier.
 19. Themethod of claim 9, wherein X_(R) and Y_(R) are selected from the groupof (X_(R), Y_(R)) pairs consisting of (5,0), (4,1), (3,2), (2,3), (1,4),and (0,5).
 20. The method of claim 13, wherein X_(R) and Y_(R) areselected from the group of (X_(R), Y_(R)) pairs consisting of (5,0),(4,1), (3,2), (2,3), (1,4), and (0,5).
 21. The method of claim 15,wherein X_(R) and Y_(R) are selected from the group of (X_(R), Y_(R))pairs consisting of (5,0), (4,1), (3,2), (2,3), (1,4), and (0,5). 22.The composition of claim 1, wherein the peptide compound comprises SEQID NO:34 [(AEEP)-(AEEP)-RRRRGTRCQVAGWGSWRSGGRLSRFPRFVNVR], wherein AEEPis [3-(2-amino-ethoxy)-ethoxy]-propanoic acid.
 23. The composition ofclaim 1, wherein the peptide compound comprises SEQ ID NO:35[(AEEP)-(AEEP)-RRRRGTRCQVAGWGSWHSGGRLSRFPRFVNVR], wherein AEEP is[3-(2-amino-ethoxy)-ethoxy]-propanoic acid.